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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, Mg 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.

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 chemiluminescenceanalysis 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.

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 T 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 β- 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.

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.

A B S T R A C TWe 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 MgCl 2 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 0.5 μM to 1 mM, 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.

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.

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