Structural basis for high-affinity recognition of aflatoxin B1 by a DNA aptamer.

The 26-mer DNA aptamer (AF26) that specifically binds aflatoxin B1 (AFB1) with nM-level high affinity is rare among hundreds of aptamers for small molecules. Despite its predicted stem-loop structure, the molecular basis of its high-affinity recognition of AFB1 remains unknown. Here, we present the first high-resolution nuclear magnetic resonance structure of AFB1-AF26 aptamer complex in solution. AFB1 binds to the 16-residue loop region of the aptamer, inducing it to fold into a compact structure through the assembly of two bulges and one hairpin structure. AFB1 is tightly enclosed within a cavity formed by the bulges and hairpin, held in a place between the G·C base pair, G·G·C triple and multiple T bases, mainly through strong π-π stacking, hydrophobic and donor atom-π interactions, respectively. We further revealed the mechanism of the aptamer in recognizing AFB1 and its analogue AFG1 with only one-atom difference and introduced a single base mutation at the binding site of the aptamer to increase the discrimination between AFB1 and AFG1 based on the structural insights. This research provides an important structural basis for understanding high-affinity recognition of the aptamer, and for further aptamer engineering, modification and applications.

Aptamer fluorescence anisotropy assays for detection of aflatoxin B1 and adenosine triphosphate using antibody to amplify signal change.

Fluorescence polarization/anisotropy (FP/FA) is an attractive technology for determining small molecules in homogeneous solution based on rotation changes of a fluorescent reporter. Binding induced conformation change is a specific property of aptamers. This property has been integrated into aptamer based FA assays for small molecules. In this work, we reported aptamer FA assays for aflatoxin B1 (AFB1) and adenosine triphosphate (ATP) by using antibody conjugated complementary DNA at the 3' end and a fluorescein (FAM)-labeled aptamer at the 5' end. The hybridization of aptamer and cDNA induced a FAM label close to the large-sized antibody, which restricts the local rotation of FAM and gives high FA signal. With the addition of target, the aptamer probe binds with the target, and the aptamer-cDNA duplex is inhibited, causing FA signal decreases. This method achieved detection of 25 pM AFB1 and 1 µM ATP, respectively. The assay is promising for application.

Immunoassay of Small Molecule Mediated by a Triply Functional DNA.

Benefiting from specific target recognition by antibodies, the immunoassay is one of the widely used assays for the detection of biologically and environmentally important small molecules in broad fields. It can be challenge to isolate small molecules from their antibody complex in an immobilization-free immunoassay with separation for the detection of small-molecule targets. Here we present an immunoassay mediated by a triply functional DNA probe. A DNA strand is dually labeled with a fluorophore and the target small molecule. This DNA probe integrates three functions, including specific binding to the antibody, signal reporting for sensitive fluorescence detection, and carrying negative charges to facilitate capillary electrophoresis (CE) separation. The binding of the probe to an antibody brings many negative charges in the complex and causes significant changes in mass-to-charge ratios, so the antibody-probe complex can be well separated from the unbound probe in CE analysis. A simple immunoassay is achieved by target competition with this DNA probe for antibody binding in CE coupled to ultrasensitive laser-induced fluorescence (LIF) detection. To show a proof of concept, we detected two model small-molecule targets, digoxin, a therapeutic drug, and ochratoxin A (OTA), an important mycotoxin for food safety. In addition, the use of two DNA probes with distinguished migration times in CE allowed the simultaneous detection of OTA and digoxin. This immunoassay provides new opportunities for wide applications.

Antibody- and aptamer-based competitive fluorescence polarization/anisotropy assays for ochratoxin A with tetramethylrhodamine-labeled ochratoxin A.

Ochratoxin A (OTA) is one of the mycotoxins that often contaminate a variety of food stuffs, and it is a potential carcinogen for humans. Taking advantage of selective affinity binding and simple, rapid, and sensitive fluorescence polarization (FP)/fluorescence anisotropy (FA) analysis, here, we report two competitive FP/FA assays for OTA using tetramethylrhodamine (TMR)-labeled OTA as a fluorescence tracer and either antibody or aptamer as an affinity ligand to recognize OTA. In the absence of OTA, the TMR-labeled OTA binds with a large-sized affinity ligand, showing a high FA value due to the slow rotation of the affinity complex. When OTA is present, OTA competes with the TMR-labeled OTA tracer in binding limited amount of affinity ligand, causing more free TMR-labeled OTA and a significant FA decrease. We found that the antibody showed a stronger affinity towards TMR-labeled OTA compared to the aptamer. The antibody-based FA assay showed higher signal changes than the aptamer based FA assay due to the larger size of antibody over aptamer. The antibody-based competitive FA assay enabled the detection of 2.4 nM OTA, while the aptamer-based FA assay also achieved a detection limit of 2.4 nM OTA at 10 °C with the help of streptavidin conjugation to increase the molecular size and to improve aptamer affinity. These two competitive FA assays were selective, showing capability for analysis in diluted red wine.

Fluorescence Anisotropy-Based Signal-Off and Signal-On Aptamer Assays Using Lissamine Rhodamine B as a Label for Ochratoxin A.

Ochratoxin A (OTA), a common mycotoxin, has attracted great concern as many foodstuffs can suffer from OTA contamination; OTA causes harmful effects on human and animals. Rapid and sensitive detection of OTA is demanded in many fields for agricultural product quality, food safety, and health. Aptamer fluorescence polarization/anisotropy (FP/FA) assays integrate advantages of nucleic acid aptamers (e.g., easy preparation, high stability, and low cost) and FP/FA analysis (e.g., high sensitivity, rapidity, simplicity, and robustness). Here, we report the preparation of lissamine rhodamine B labeled OTA and developed competitive aptamer fluorescence anisotropy (FA) assays for OTA with signal-off or signal-on responses by using this fluorescently labeled probe. In the signal-off FA assay, the binding between the fluorescent probe and aptamer gave a large FA signal due to molecular volume increase, and the fluorescent probe was displaced from the aptamer in the presence of OTA target, causing FA to decrease. To further enhance the FA change in the signal-off assay, large-sized streptavidin was conjugated on the aptamer, and this assay allowed for a detection limit of 2.5 nM and a more remarkable FA decrease. Furthermore, we found that the fluorescent probe could interact with Tween 20, which caused the fluorescent probe to show a higher FA value than that of the aptamer-fluorescent probe complex. A signal-on FA assay was achieved in the binding buffer containing 0.1% Tween 20, with a detection limit of 10 nM. Signal-off and signal-on FA methods both were selective and enabled detection of OTA spiked in red wine samples, showing capability for target analysis in complex sample matrix.

An immunoassay for ochratoxin A using tetramethylrhodamine-labeled ochratoxin A as a probe based on a binding-induced change in fluorescence intensity.

Ochratoxin A (OTA) is a mycotoxin that can cause health risks to human/animal health. Contamination by OTA can occur in various foods and agricultural products, so sensitive and rapid detection of OTA is crucial. We describe a simple and sensitive fluorescence immunoassay for OTA using tetramethylrhodamine (TMR)-labeled OTA as a fluorescent probe. We conjugated tetramethylrhodamine to OTA through a covalent reaction, and obtained three TMR-OTA isomer probes after purification by high-performance liquid chromatography. All of the fluorescent probes showed high binding affinity to the anti-OTA antibody. Binding of the TMR-OTA probe to the antibody induced strong fluorescence of TMR-OTA due to the possible change in the local environment of TMR caused by affinity binding. In the presence of OTA, the OTA target competitively displaced the bound TMR-OTA probe from the antibody, causing a decrease in fluorescence. Measuring the change in fluorescence enabled rapid detection of OTA. This method was selective and allowed the detection of 1 nM OTA, showing potential for rapid OTA analysis in applications.

Fluorometric determination of aflatoxin B1 using a labeled aptamer and gold nanoparticles modified with a complementary sequence acting as a quencher.

A fluorometric aptamer based assay is described for rapid and sensitive detection of aflatoxin B1 (AFB1). It is making use of a fluorescein (FAM) labeled anti-AFB1 aptamer and complementary DNA-modified gold nanoparticles (GNPs). In the absence of AFB1, the FAM-labeled aptamers hybridize with complementary DNA strands that were covalently immobilized on GNPs. This results in quenching of the green fluorescence (with excitation/emission peaks at 485/525 nm). In the presence of AFB1, the aptamer probe binds AFB1 and is released from the GNPs. Hence, fluorescence is restored. Under optimized conditions, AFB1 in the concentration range from 61 pM to 4.0 µM can be detected, and the detection limit is 61 pM. This assay is highly selective for AFB1. It was applied to the determination of AFB1 spiked into 50-fold diluted wine and 20-fold diluted beer. Graphical abstract Schematic presentation of fluorometric detection of AFB1 using a fluorescein (FAM) labeled anti-AFB1 aptamer and complementary DNA-modified gold nanoparticles (GNPs).

A signal-on electrochemical aptasensor for rapid detection of aflatoxin B1 based on competition with complementary DNA.

Aflatoxin B1 (AFB1) is the most toxic mycotoxin, causing harmful effects on human and animal health, and the rapid and sensitive detection of AFB1 is highly demanded. We developed a simple electrochemical aptasensor achieving rapid detection of aflatoxin B1 (AFB1). A short anti-AFB1 aptamer having a methylene blue (MB) redox tag at the 3'-end was immobilized on the surface of a gold electrode. In the absence of AFB1, a complementary DNA (cDNA) strand hybridized with the MB-labeled aptamer, causing MB apart from the electrode surface and low current of MB. In the presence of AFB1, AFB1 competed with the cDNA in the binding to the MB-labeled aptamer, and the aptamer-AFB1 binding caused formation of a hairpin structure, making the MB close to the electrode surface and current of MB increase. Under optimized conditions, we achieved detection of AFB1 over dynamic concentration range of 2 nM-4 µM by using this signal-on electrochemical aptasensor. This method only required a simple 5-min incubation of sample solution prior to rapid electrochemical sensing, more rapid than other electrochemical aptasensors. The sensor could be well regenerated and reused. This sensor allowed to detect AFB1 spiked in 20-fold diluted beer and 50-fold diluted white wine, respectively. It shows potential for detection of AFB1 in wide applications.

An aptamer assay for aflatoxin B1 detection using Mg2+ mediated free zone capillary electrophoresis coupled with laser induced fluorescence.

We described an aptamer based and Mg2+ mediated free zone capillary electrophoresis-laser induced fluorescence (CE-LIF) assay for aflatoxin B1 (AFB1) detection. This CE-LIF assay applied an anti-AFB1 aptamer with a single fluorescein (FAM) label at 5' end and a short complementary DNA (cDNA). In the absence of AFB1, the cDNA hybridized with the aptamer probe and formed a duplex DNA. The use of running buffer containing MgCl2 allowed good isolation of the duplex DNA from the single stranded DNA in CE. We found introducing a biotin label on the cDNA further improved the isolation. When AFB1 existed in sample solution, the aptamer probe bound with AFB1, dissociating from the duplex DNA. Thus, the duplex DNA peak decreased, while the aptamer probe peak increased during CE-LIF analysis. We achieved detection of AFB1 by measuring the aptamer probe peak. The length of cDNA, the ratio of aptamer to cDNA, and the concentration of MgCl2 in sample buffer and separation buffer had great effect on the aptamer based CE-LIF assay. Under optimized conditions, the detection limit of AFB1 was 0.2 nM, and the dynamic range was from 0.2 nM to 500 nM. Limit of quantitation was 0.5 nM. This CE-LIF assay enabled detection of AFB1 spiked in diluted human serum, diluted human urine, and corn flour samples. This assay exhibits potential for wide application as it integrates the rapidity, high sensitivity, low sample consumption of CE-LIF analysis and the strengths of aptamer.

Aptamer Structure Switch Fluorescence Anisotropy Assay for Small Molecules Using Streptavidin as an Effective Signal Amplifier Based on Proximity Effect.

Fluorescence polarization/anisotropy (FP/FA) approaches are appealing for targets sensing in homogeneous solution due to simplicity, reproducibility and sensitivity. Taking advantage of aptamers, aptamer structure switch FA methods are unique for small molecule detection based on the competition between aptamer-target binding and the hybridization of aptamer and complementary DNA (cDNA). However, usually small FA change is generated in these aptamer assays that only rely on size change caused by hybridization of an oligonucleotide because of the rapid local rotation of fluorophores and small mass change. Here we describe a simple and general aptamer structure switch FA assay for small molecules by employing a large-sized streptavidin (SA) as an effective signal amplifier based on proximity effect to reduce local rotation of fluorophore. In this design, the SA-labeled cDNA hybridizes with fluorescein (FAM)-labeled aptamer, drawing FAM close to SA and bringing a much higher FA value due to restricted local rotation of FAM. Small molecule-aptamer probe binding causes displacement of the SA-labeled cDNA and great decrease of FA. The closeness of SA to FAM in the duplex is key for this proposed strategy to produce large FA changes in target detection. Our method enabled to detect 60 pM aflatoxin B1 (AFB1), 1 nM ochratoxin A (OTA), and 0.5 µM adenosine triphosphate (ATP), respectively. This aptamer FA method combines the merits of aptamers and FA analysis, and it is promising in applications of detection of small molecules with good sensitivity.

Aptamer-Structure Switch Coupled with Horseradish Peroxidase Labeling on a Microplate for the Sensitive Detection of Small Molecules.

Detection of small molecules with good sensitivity, high throughput, simplicity, and generality using aptamers is desired but still remains challenging. We described an aptamer-structure-switch assay coupled with horseradish peroxidase (HRP) labeling on microplates for sensitive absorbance and chemiluminescence detection of small molecules. This assay relies on competition for affinity binding to a limited HRP-labeled aptamer between small-molecule targets and immobilized short DNA strands complementary to the aptamer (cDNA) on a microplate. In the absence of targets, the HRP-labeled aptamer hybridizes with the cDNA on the microplate, and HRP catalyzes substrate into product, generating absorbance or chemiluminescence signals. The binding of small-molecule targets to aptamers causes displacement of HRP-labeled aptamers from the cDNA and signal decrease. In chemiluminescence-analysis mode, the assay achieved detection of aflatoxin B1 (AFB1), ochratoxin A (OTA), and adenosine triphosphate (ATP) with detection limits of 10 pM, 20 pM, and 20 nM, respectively. This assay does not require enzyme-labeled small molecules or the conjugation of small molecules on solid phase. HRP, as an enzyme label, here allows for easily obtainable and highly active signal amplification. This microplate assay is rapid and promising for high-throughput analysis. It shows potential for wide applications in the detection of small molecules.

Development of aptamer fluorescent switch assay for aflatoxin B1 by using fluorescein-labeled aptamer and black hole quencher 1-labeled complementary DNA.

Aflatoxin B1 (AFB1) is one of the most toxic mycotoxins and draws great concern in health and food safety. A DNA aptamer against AFB1 having a stem-loop structure shows high binding affinity to AFB1 and promise in assay development for AFB1 detection. Based on the structure-switching property of the aptamer, we report an aptamer fluorescence assay for AFB1 detection. Aptamer with fluorescein (FAM) label at 5' end was used as affinity ligand, while its short complementary DNA (cDNA) with BHQ1 (black hole quencher 1) label at 3' end was used as a quencher. In the absence of AFB1, FAM-labeled aptamer hybridized with BHQ1-labeled cDNA, forming a duplex of cDNA and aptamer, resulting in fluorescence quenching of FAM. When AFB1 bound with aptamer, the BHQ1-labeled cDNA was displaced from aptamer, causing fluorescence restoration of FAM. We tested a series of FAM-labeled aptamers and BHQ1-labeled cDNAs with different lengths. The lengths of the aptamer stem and the cDNA, Mg2+ in binding buffer, and temperature had significant influence on the performance of the assay. Under optimized conditions, we achieved sensitive detection of AFB1 by using a 29-mer FAM-labeled aptamer and a 14-mer BHQ1-labeled cDNA, and the detection limit of AFB1 reached 0.2 nM. The maximum fluorescence recovery rate of FAM-labeled aptamer caused by AFB1 was about 69-fold. This method enabled the detection of AFB1 in complex sample matrix, e.g., diluted wine samples and maize flour samples. This aptamer-based fluorescent assay for AFB1 determination shows potential for broad applications. Graphical abstract ᅟ.

Enhancing the Affinity of Anti-Human α-Thrombin 15-mer DNA Aptamer and Anti-Immunoglobulin E Aptamer by PolyT Extension.

Aptamer affinity capillary electrophoresis-laser-induced fluorescence (CE-LIF) for protein detection takes advantage of aptamers for their ease of synthesis and labeling, small size, and having many negative charges. Its success relies on the high binding affinity of aptamers. One 15-mer DNA aptamer (5'-GGT TGG TGT GGT TGG-3', Apt15) shows desirable specificity for human α-thrombin, an important enzyme with multiple functions in blood. However, Apt15 has weak binding affinity, and the use of Apt15 in affinity CE-LIF analysis remains challenging. Here we reported that extension of Apt15 at the 3'-end with a polyT tail having length of 18 T or longer significantly enhanced its affinity and enabled a well-isolated and stable peak for thrombin-aptamer complex in affinity CE. It was likely that the improvement of binding affinity resulted from double binding, an additional interaction of the polyT tail with thrombin in addition to the Apt15 section binding to thrombin. With dye-labeled Apt15 having a T25 tail, we achieved detection of thrombin at concentrations as low as 0.1 nM by affinity CE-LIF. This aptamer probe specifically bound to human α-thrombin, showing negligible affinity for human ß- and γ-thrombin, which are proteolyzed derivatives of human alpha α-thrombin and share similar structure. This strategy of adding a polyT extension also enhanced the binding affinity of anti-immunoglobulin E aptamer in CE-LIF analysis, showing that the affinity enhancement approach is not limited to the thrombin-binding aptamer and has potential for more applications in bioanalysis.

Competitive fluorescence anisotropy/polarization assay for ATP using aptamer as affinity ligand and dye-labeled ATP as fluorescence tracer.

We developed an aptamer-based competitive fluorescence anisotropy (FA)/fluorescence polarization (FP) assay for adenosine triphosphate (ATP). Different from the traditional fluorescence polarization immunoassays for small molecules, here DNA aptamer against ATP was used as affinity ligand, and tetramethylrhodamine (TMR) labeled ATP served as fluorescent tracer. The binding between TMR-labeled ATP and aptamer gave large FA due to molecular volume increase and restricted rotation of the dye-labeled ATP. When ATP was added in solution, ATP competitively displaced the TMR-labeled ATP from aptamer affinity complex, causing decrease of FA of TMR-labeled ATP. The buffer containing MgCl2 and incubation at low temperature were preferred for large FA change in the FA assay. The FA change was further enhanced in this competitive FA assay by increasing the molecular weight of aptamer through extension of aptamer sequences or conjugating streptavidin protein on aptamer. This method allowed for the detection of ATP in the range from 0.5µM to 1mM, generating the maximum FA change about 0.187 (corresponding maximum FP change about 0.242). The detection of ATP spiked in diluted urine or serum sample was achieved, showing capability for analysis in complex sample matrix. This assay also enabled the detection of the analogues of ATP, e.g. adenosine, adenosine monophosphate (AMP), and adenosine diphosphate (ADP) with similar sensitivity. This aptamer-based competitive FA assay takes advantages of aptamer in ease of synthesis, good thermal stability, and facile modulating the molecular mass of aptamer.