Aflatoxin B1 Aptamer Research Articles

Ratiometric electrochemical and impedimetric dual-mode aptasensor based on thionine-functionalized Ti3C2Tx MXene/Pt and Au nanoparticle composites for reliable detection of aflatoxin B1

Rapid and reliable detection of aflatoxin B1 (AFB1) is critical in public health, food safety and environmental control. Herein, a ratiometric electrochemical (EC) and electrochemical impedance spectroscopy (EIS) dual-mode aptasensing platform based on thionine-functionalized Ti3C2Tx MXene/Pt and Au nanoparticle composites (MXene@Thi/PtAuNPs) was constructed for accurate and reliable detection of AFB1. The MXene@Thi/PtAuNPs composites were prepared by a facile and mild approach. The electroactive molecule Thi embedded in MXene could not only inhibit the aggregation of MXene nanosheets, but also act as an internal reference probe to form a ratiometric sensing strategy. The decoration of Pt and Au nanoparticles can further effectively enhance the conductivity and catalytic activity of the composites. The MXene@Thi/PtAuNPs composites were then served as a support to sequentially assemble hairpin DNA (hDNA) and ferrocene-labelled AFB1 aptamer (Fc-Apt) to construct the dual-mode sensor. The developed dual-mode sensor with both ratiometric EC detection (based on IThi/IFc) and EIS detection (based on Ret) can mutually verify the detection results obtained by two modes, offering more accurate and reliable results, which enabled sensitive and reliable AFB1 detection in the concentration range of 10.0 pg mL−1-30.0 ng mL−1 (ratiometric EC mode) and 3.0 pg mL−1-30.0 ng mL−1 (EIS mode), respectively. The sensing platform also demonstrated favorable selectivity, reproducibility and stability, and satisfactory applicability in corn sample, showing great potential in the sensing of mycotoxins in agricultural products.

DNA tetrahedral scaffold based one-step ratiometric electrochemical detection of aflatoxin B1 in tobacco samples

To effectively monitor Aflatoxin B1 (AFB1) in tobacco samples, a ratiometric electrochemical biosensor based on DNA tetrahedral scaffold (DTS) and AuNPs/chitosan (CS) modified electrode has been successfully fabricated. The ultra-thin AuNPs not only present a larger specific sensing surface, but also can graft more DTS onto the surface of the electrode. The DTS is directly grated with two strands of AFB1 aptamer, which leading to a facile one-step detection of AFB1. In the presence of AFB1, the current of methylene blue (MB) keeps stable, while the current of ferrocene (Fc) decreases since the target takes away the Fc-labeled aptamer from the electrode surface. The fabricated biosensor can monitor AFB1 in the linear range of 0.01–1000 pg·mL−1 with the detection limit of (LOD) 2.6 fg·mL−1. More importantly, the biosensor performs well in the detection of AFB1 in tobacco samples with the LOD of 2.86 fg·mL−1. The developed ratiometric electrochemical biosensor displays high selectivity, good stability and high potential in tobacco sample’s AFB1 detection.

A highly sensitive fluorescence biosensor for aflatoxins B1 detection based on polydiacetylene liposomes combined with exonuclease III-assisted recycling amplification.

Afluorescence biosensor for determination of aflatoxin B1 (AFB1)based on polydiacetylene (PDA) liposomes and exonuclease III (EXO III)-assisted recycling amplification was developed. The AFB1 aptamer partially hybridizes with complementary DNA (cDNA), which is released upon recognition of AFB1 by the aptamer. Subsequently, the cDNA hybridizes with hairpin H to form double-stranded DNA that undergoes digestion by EXO III, resulting in the cyclic release of cDNA and generation of capture DNA for further reaction. The capture DNA then hybridizes with probe modified on PDA liposomes, leading to aggregation of liposomes and subsequent fluorescence production. This strategy exhibited a limit of detection of 0.18 ng/mL within the linear range 1-100 ng/mL with adetermination coefficient > 0.99. The recovery ranged from 92.81 to 106.45%, with relative standard deviations (RSD) between 1.73 and 4.26%, for corn, brown rice, peanut butter, and wheatsamples. The stability, accuracy, and specificity of the method demonstrated the applicability for realsampleanalysis.

Detection of AFB1 in corn by MXene paper‐based unlabeled aptasensor

AbstractIn this work, a paper‐based electrochemical aptamer sensor was developed for the detection of aflatoxin B1 (AFB1) using a combination of MXene–Ti3C2Tx and nucleic acid aptamers. The prepared single‐layer or few‐layer MXene suspension is suction‐filtered onto MXene paper, which is cut to prepare MXene electrodes. To accomplish AFB1 specific detection, an amino‐labeled AFB1 aptamer is mounted on the surface of the carboxy‐functionalized MXene electrode. When AFB1 is present, it particularly binds to the aptamer to form a 3D structure, reducing the efficiency of electron transmission on the sensor surface. The difference in impedance signal change at the electrode/electrolyte interface is used to quantify AFB1. The results indicated that the detection range is 0.05–100 ng/mL, the detection limit is 0.04 ng/mL, and the recovery rate of AFB1 in corn samples is 97.8%–111.52% with the optimal detection conditions. The MXene paper‐based label‐free aptasensor is versatile and can detect different targets by simply swapping out the aptamers of different targets. The sensor also has a wide range of applications in food analysis and environmental testing.Practical applicationsA paper‐based electrochemical aptamer sensor was developed to detect aflatoxin B1 using a combination of MXene–Ti3C2Tx and nucleic acid aptamers.The design is based on the preparation of MXene electrodes by pumping and filtering monolayer or multilayer MXene suspensions onto MXene paper and cutting.The MXene paper‐based label‐free aptamer sensor was designed to be versatile, allowing the detection of different targets by simply replacing the aptamer with one from a different targets.

A biosensor method based on surfactant-mediated surface droplet evaporation for the detection of Aflatoxin B1

Aflatoxin B1 (AFB1) is a common mycotoxin that is of significant global concern due to its impact on food safety. Herein, we innovatively develop a sensing platform to detect AFB1 based on evaporation of surfactant solutions on the hydrophobic surface, resulting in dried patterns with varied sizes. The surfactant CTAB solution produces a relatively large dried pattern due to the surface wetting. However, the reduction in the dried pattern size is found when the mixture of CTAB and AFB1 aptamer is tested, because the formation of CTAB/aptamer complex. Moreover, the dried pattern size of the mixture of CTAB, aptamer, and AFB1 increases due to the specific binding of AFB1 to its aptamer. Using this innovative strategy, the AFB1 detection can be fulfilled with a detection limit of 0.77 pg/mL. As a simple, convenient, inexpensive, and label-free method, the surfactant-mediated surface droplet evaporation-based biosensor is very promising for various potential applications.

Simultaneous Detection of Ochratoxin A and Aflatoxin B1 Based on Stable Tuning Fork-shaped DNA Fluorescent Aptasensor.

Tuning fork, consisting of two fork arms and a fork handle, has a stable and rigid structure. Inspired by this structure, a tuning fork-shaped DNA (TF-DNA) fluorescence aptasensor was constructed to detect ochratoxin A (OTA) and aflatoxin B1 (AFB1). A TF-DNA double-stranded structure capable of attaching both OTA aptamer labeled with the FAM fluorescent group (FAM-Apt) and AFB1 aptamer labeled with the ROX fluorescent group (ROX-Apt) was designed and linked to magnetic beads. This TF-DNA double-stranded structure can provide a stable platform for dual-target detection. In the presence of OTA and AFB1, FAM-Apt and ROX-Apt preferentially bound to them and detached from the TF-DNA double-stranded structure. Dual-signal fluorescent probes were collected from the supernatant by magnetic separation, and achieved fluorescence enhancement at 520nm and 607nm, respectively. The linear ranges are 0.05ng/mL to 100ng/mL for OTA and 0.1ng/mL to 100ng/mL for AFB1, and the detection limits are 0.015ng/mL and 0.045ng/mL, respectively. The developed sensor has the advantages of simple and fast preparation, good specificity and reproducibility, which is promising for the simultaneous determination of multiple hazardous substances in food.

Polycarboxyl ionic liquid functionalized Yb-MOFs nanoballs based dual-wavelength responsive photoelectrochemical aptasensor for the simultaneous determination of AFB1 and OTA

Developing an accurate and precise approach for the simultaneous detection of ochratoxin A (OTA) and aflatoxin B1 (AFB1) is significant for food safety surveillance. Herein, a photoelectrochemical sensing platform was constructed based on polycarboxylic ionic liquid functionalized metal-organic framework integrated with gold nanoparticles (Yb-MOFs@AuNPs). Sulfhydryl functionalized hairpin DNA (hDNA) was immobilized on a Yb-MOFs@AuNPs modified glassy carbon electrode (GCE) surface through Au-S bond. After blocking residual active binding sites with BSA, gold nanoparticles-labeled AFB1 aptamer (AuNPs-Apt 1) and gold nanorods-labeled OTA aptamer (AuNRs-Apt 2) were introduced to construct a photoelectrochemical aptasensor for the simultaneous determination of AFB1 and OTA. Due to the surface plasmon resonance effect and the nanometer size effect of gold nanomaterials, the photoelectrochemical aptasensor can output photocurrent responses as being excited with different wavelengths at 520 nm and 808 nm, respectively. When the AFB1 and OTA concentration in the range of 0.001–50.0 ng mL−1, a good linear relationship between the photocurrent difference (ΔI) before and after recognizing targets and the logarithm of AFB1 or OTA concentration was obtained. The detection limits for AFB1 and OTA were 0.40 pg mL−1 and 0.19 pg mL−1, respectively. AFB1 and OTA in corn samples were detected simultaneously by the photoelectrochemical aptasensor.

Regenerable ratiometric aptasensor based on electro-oxidation conducted host-guest dissociation for aflatoxin B1 detection in grains

Aflatoxin B1 (AFB1) leads to considerable pollution to grains and causes harmful effects on human health. Herein, we present a regenerable AFB1 aptasensor, based on electro-oxidation conducted host-guest dissociation for natural AFB1 detection. Molybdenum disulfide-Au nanobipyramids (MoS2-AuNBPs) serve as signal carriers and are equipped with indicative thionine (Thi) and semi-complementary chains to AFB1 aptamer (ssDNA). The electrode surface is modified with gold nanoparticles (AuNPs) and β-cyclodextrin (β-CD). Aptamer-ferrocene (Apt-Fc) can be recognized in cavity of β-CD through host-guest interaction. The signal probe, self-assembled with AFB1 Apt via hybridization, can be easily expelled by AFB1, bringing a ratiometric readout ΔIThi/IFc. Particularly, regeneration process is realized with synergy of AFB1-conducted Apt-ssDNA dissociation and electro-oxidation conducted host (β-CD)-guest (Fc) dissociation. Modifications and regeneration behaviors are morphologically and electrochemically validated. Regenerations are dependably performed in repetitive cycles (N = 7), which exhibit considerable performance in analyses. Electro-oxidation time is experimentally optimized (6 min). This method exhibits wide range from 1.0 pg/mL to 80.0 ng/mL, and limit of detection is 5.0 × 10−1 pg/mL, to target AFB1. During natural AFB1 determinations, proposed readouts are validated with certified HPLC and recovery test. Relative errors and recovery rates are calculated as 2.04–7.22% and 94.41–106.54%, respectively.

Self-assembled programmable DNA nanoflower for in situ synthesis of gold nanoclusters and integration with Mn-MOF to sensitively detect AFB1

Aflatoxin B1 (AFB1) is a potent carcinogenic mycotoxin that is susceptible to contamination of agricultural products and can cause chronic effects on humans. Thus, the establishment of highly sensitive and efficient AFB1 detection method is crucial to ensure food safety and human health. Here, we developed programmable DNA nanoflowers (DNF) with dual functionality for in situ synthesis of gold nanoclusters (AuNCs) and DNA recognition. The AuNCs synthesized using DNF as template (DNF@AuNCs) possessed good fluorescence properties and stability, and were applied as stable signal probes for constructing sensitive aptasensor. DNF completed the transition from bud to blooming flower, shifting its morphology from 3D to 2D structure during the in-situ synthesis of AuNCs, leading to the exposure of more template sequences. The designed hairpins DNA were subsequently used for local catalytic hairpin assembly (LCHA) on the surface of Mn-MOF. A substantial number of amplified products H2-H3 was obtained by conformational conversion, which was complementary to the sequence of DNF. Upon hybridization of H2-H3 with DNF@AuNCs, the fluorescence of AuNCs is quenched by adjacent guanine-rich (G-rich) base at the 3′ end of H3. The switch of LCHA is controlled by the conformational change of H1 which contains the AFB1 aptamer sequence, thus enabling precise detection of AFB1. The DNF@AuNCs and Mn-MOF-based fluorescence aptasensor achieved a detection limit of 7 pg/mL for AFB1, with a detection range of 0.01–200 ng/mL. The aptasensor also exhibits high selectivity, reproducibility, and stability, making it prospective for food safety monitoring.

A novel highly active AgMOF-based silver single-atom catalyst and its application to the aptamer SERS/RRS for the determination of aflatoxin B1

A novel highly active silver single-atom catalyst (AgSAC) was prepared by a microwave-assisted solvothermal method using silver covalent organic frameworks (AgMOF) as precursors. It was characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), infrared (IR), and surface-enhanced Raman scattering (SERS). The experiment found that AgSAC has excellent catalytic performance and can heavily catalyze the nano-reaction of chloroauric acid-malic acid (HAuCl4–H2Mi) to generate gold nanoparticles (AuNPs). The produced AuNPs have strong SERS, resonance Rayleigh scattering (RRS) and surface plasmon resonance absorption (Abs) signals. Aflatoxin B1 aptamer (AptAFB1) can be adsorbed to the surface of AgSAC through electrostatic interaction, to reduce the catalytic activity of AgSAC and the SERS/RRS/Abs signal of the system. When the target molecule (AFB1) was added, it will specifically bind to AptAFB1 and release AgSAC, restoring the catalytic activity of AgSAC, thereby restoring the SERS/RRS/Abs signal of the system. Based on this, a simple and sensitive aptamer sensing analysis platform for trace AFB1 was established, and a reasonable catalytic amplification mechanism of AgSAC was proposed. The SERS method exhibited the highest sensitivity, with a linear range of 0.005–0.225 μg/L and a detection limit of 0.002 μg/L.

Nitrogen-doped carbon dot and DNA tetrahedron nanostructure based electrochemiluminescence aptasensor for AFB1 detection

An electrochemiluminescence (ECL) aptasensor with aptamer as recognition element, N-doped carbon dots (N-CDs) as luminophore, and DNA tetrahedral nanostructure (DTN) as carrier to loading abundant N-CDs for signal amplification was developed for ultra-sensitive aflatoxin B1 (AFB1) detection. The complementary DNA (cDNA) was firstly immobilized on the Au electrode (AuE) surface to hybridize with the AFB1 aptamer. In the presence of AFB1, it would competitively with cDNA for binding the aptamer, causing the exposure of cDNA. DTN modified with AFB1 aptamer on top and N-CDs on the four vertices would combine with cDNA onto the AuE surface to generate ECL signals due to electron transfer between co-reactants (K2S2O8) and N-CDs, which was positively related to the concentration of AFB1. Due to the high specificity of aptamer, the outstanding ECL performance of N-CDs and the enhanced signal output of DTN/N-CDs probes, the as-constructed ECL aptasensor realized excellent sensitivity with a low detection limit of 77 pg/mL and a wide linear concentration of 1.0 × 10−1-1.0 × 104 ng/mL for AFB1, as well as fascinating accuracy with acceptable recoveries of 89.54–111.0% in the spiked Coix seed matrices. Successful application for AFB1 detection in actual samples confirmed the extensive practicability of the ECL aptasensor, providing a universal platform for more trace contaminants by altering the corresponding aptamers.

A novel Recjf Exo signal amplification strategy based on bioinformatics-assisted truncated aptamer for efficient fluorescence detection of AFB1

Aflatoxin B1 (AFB1) is a typical mycotoxin that signifacntly endangers public health and economy. In this study, we systematically studied the interaction of aptamers with AFB1 using circular dichroism, molecular dynamics, molecular docking, and fluorescence analysis. The truncated sequence aptamers were screened using molecular docking. We successfully obtained the AFB1 aptamer with higher affinity and its truncated form was enhanced by 5.2-fold compared to the initial AFB1 aptamer. In addition, for rapid detection of AFB1, we designed a fluorescent nano-adaptor sensing platform using RecJf exonuclease signal amplification strategy based on the optimal aptamer. The aptasensor showed satisfactory sensitivity towards AFB1 with a linear detection range of 1–400 ng/mL and a detection limit of 0.57 ng/mL. The aptasensor was successfully applied to the determination of AFB1 in soybean oil and corn oil with recoveries of 91.02 %–106.59 % and 87.39 %–110.61 %, respectively. The successful application of the AFB1 aptasensor, developed through bioinformatics truncation of the aptamer, provides a novel approach to creating a cost-effective, eco-friendly, and rapid aptamer sensing platform.

Single-Walled Carbon Nanohorn-Based Fluorescence Energy Resonance Transfer Aptasensor Platform for the Detection of Aflatoxin B1.

Aflatoxin B1 (AFB1) is one of the most contaminated fungal toxins worldwide and is prone to cause serious economic losses, food insecurity, and health hazards to humans. The rapid, on-site, and economical method for AFB1 detection is need of the day. In this study, an AFB1 aptamer (AFB1-Apt) sensing platform was established for the detection of AFB1. Fluorescent moiety (FAM)-modified aptamers were used for fluorescence response and quenching, based on the adsorption quenching function of single-walled carbon nanohorns (SWCNHs). Basically, in our constructed sensing platform, the AFB1 specifically binds to AFB1-Apt, making a stable complex. This complex with fluorophore resists to be adsorbed by SWCNHs, thus prevent SWCNHs from quenching of fluorscence, resulting in a fluorescence response. This designed sensing strategy was highly selective with a good linear response in the range of 10-100 ng/mL and a low detection limit of 4.1 ng/mL. The practicality of this sensing strategy was verified by using successful spiking experiments on real samples of soybean oil and comparison with the enzyme-linked immunosorbent assay (ELISA) method.

Simultaneous measurement of ochratoxin A and aflatoxin B1 using a duplexed-electrochemical aptasensor based on carbon nanodots decorated with gold nanoparticles and two redox probes hemin@HKUST-1 and ferrocene@HKUST-1

An electrochemical aptasensor was developed for the simultaneous determination of ochratoxin A (OTA) and aflatoxin B1 (AFB1). Two compounds hemin@HKUST-1 and ferrocene@HKUST-1 were synthesized by encapsulating hemin and ferrocene inside the MOF made by copper nodes and trimesic acid, known as HKUST-1 MOF, and then bind to complementary DNA sequences of OTA aptamer (cDNA1) and AFB1 aptamer (cDNA2), respectively. Then, the hemin@HKUST-1/cDNA1 and ferrocene@HKUST-1/cDNA2 bioconjugates were deposited on the glassy carbon electrode (GCE) modified with carbon nanodots decorated with gold nanoparticles (AuNPs-CNDs). In the absence of the two mycotoxins, the current response related to the electroactive labels of hemin and ferrocene in the discussed bioconjugates have been measured at two separate potentials simultaneously by the differential pulse voltammetry technique (DPV). Using the described aptasensor, it is possible to measure both OTA and AFB1 mycotoxins in the linear concentration range of 1.0 × 10−2 to 100.0 ng mL−1. Also, the detection limits of OTA and AFB1 were 4.3 × 10−3 ng mL−1 and 5.2 × 10−3 ng mL−1, respectively. Finally, the ability of the proposed duplex aptasensor for the simultaneous measurement of OTA and AFB1 in a corn flour sample was investigated and completely satisfactory results were achieved.

A gold nanoflower based dual mode aptasensor for aflatoxin B1 detection using SERS and fluorescence effect simultaneously

Aflatoxin B1 (AFB1) is usually the major aflatoxin produced by toxigenic strains and has been identified the most potent natural carcinogen. Here, a SERS/fluorescence dual-mode nanosensor has been designed while gold nanoflowers (AuNFs) was used as substrate for the detection of AFB1. AuNFs exhibited excellent SERS enhancement effect as well as the good fluorescence quenching effect which made the dual signal detection possible. First, the surface of AuNFs was modified with AFB1 aptamer via Au-SH group. Then, the complementary sequence functionalized with Cy5 (the signal molecule) was attached to AuNFs based on the base complementary pairing principle. On this case, Cy5 was close to AuNFs, the SERS intensity was greatly enhanced and the fluorescence intensity was quenched. After incubation with AFB1, the aptamer was preferentially combined to its target AFB1. Thus, the complementary sequence detached from AuNFs which caused the SERS intensity of Cy5 decreased while its fluorescence effect recovered. Then, the quantitative detection was realized with two optical properties. The LOD was calculated to be 0.03 ng/mL. It was a convenient and fast detection method which expanded the application of nanomaterials based multi-signal simultaneous detection.

From food toxins to biomarkers: Multiplexed detection of aflatoxin B1 and aflatoxin M1 in milk and human serum using PEGylated ternary transition metal sulfides

Aflatoxins (AF) are fungal toxins which not only contaminate food, but also adversely impact human health if consumed and serve as biomarkers for deadly diseases like liver cancer. Therefore, there is a strong need to develop ultra-sensitive AF detection method in physiological environments. In the present study, ternary transition metal sulfides single crystals (Mn0.02Ta3S6 and Fe0.65Ta3S6) were grown, exfoliated to thin layered nanosheets (NSs) followed by polyethylene-glycol modification (PEG@NSs). High fluorescence quenching abilities towards the aptamers for AF biosensing were demonstrated using PEGylated NSs. It has been observed that PEGylated NSs improves the detection sensitivity ∼ 248 times better than non-PEGylated NSs. Subsequently, multiplexed detections of aflatoxin B1 (AFB1) and M1 (AFM1) in diverse mediums including PBS buffer (1 ×), milk and human serum were performed by employing PEG@Mn0.02Ta3S6 NSs with TAMRA dye-labelled AFB1 aptamer and FAM dye-labelled AFM1 aptamer, respectively. The fluorescence intensity of designed sensor exhibited ultrasensitive detections (∼ pM) and a wide linear range (≥ 5 orders). Hence, the present study can serve as a unique platform for facile, ultra-sensitive, selective, cost-effective and quick multiplexed detection of food toxins and disease biomarkers in complex-matrix.

Optical approaches for the simple and sensitive detection of aflatoxin B1 via a liquid crystal aptasensor

ABSTRACT We report a direct observation method for the detection of aflatoxin B1 (AFB1), utilising the orientational properties of liquid crystals (LCs). The imaging principle is based on the orientation transition of an LC film, which can be influenced by an aptamer and AFB1. AFB1 is captured by aptamers fixed on a substrate, which causes a conformational change of the aptamers that induces a significant morphological change of the substrate. The AFB1–aptamer interaction induces an orientation transition of LCs from a homeotropic to a planar state at the LC – substrate interface, leading to changes from ‘dark’ to ‘bright’ responses that can be easily seen through crossed polarisers. The developed sensor exhibits high specificity for AFB1 determination and has a low detection limit of 5 fM. The sensor allows AFB1 to be determined in complex samples such as corn and rice powder. The sensing method is highly selective and sensitive for other toxins and common molecules in food. Also, the sensor is stable and easily regenerated. The proposed LC-based aptasensor is a simple, rapid, and convenient platform for label-free detection of AFB1 in real samples, and the design strategy may also provide a novel way for detecting multiple analytes.

Structural insights into the AFB1 aptamer coupled with a rationally designed CRISPR/Cas12a-Exo III aptasensor for AFB1 detection

Aflatoxin B1 (AFB1) is a typical food contaminant. A truncated DNA aptamer of AFB1 was reported by our team in previous work. However, the recognition mechanism between aptamer and AFB1 was lacking, which was crucial for the design of related aptasensor. Herein, the binding of aptamer to AFB1 was systematically studied and found that it was an exothermic process and the conformation of aptamer changed during the recognition process. Loop bases in the secondary structure of aptamer formed a special binding pocket to recognize AFB1. Van der Waals and electrostatic interaction were the main driving forces. By blocking the stem bases guided by the structural investigation, a rationally designed CRISPR/Cas12a-Exo III aptasensor for AFB1 detection was constructed, and the sensitivity was improved by target recycling. Under optimal conditions, the linear detection range for AFB1 was 0.01–20 ng/mL, and AFB1 was accurately determined in corn and wheat samples. This work laid a theoretical foundation for the design of AFB1 aptasensor, and the developed detection model came up with new ideas for the development of CRISPR/Cas12a-based aptasensor.

Dual-signal output fluorescent aptasensor based on DNA programmability and gold nanoflowers for multiple mycotoxins detection.

Herein, a dual-signal output fluorescent aptamer sensor was constructed for the simultaneous detection of aflatoxin B1 (AFB1) and ochratoxin A (OTA) using the specific recognition ability of aptamers and the programmability of DNA. A functional capture probe (cDNA) was designed with the black hole quenching motif BHQ1 labeled at the 5' end and biotin (bio) labeled at the 3' end. The fluorescent dye Cy3-labeled aflatoxin B1 aptamer (AFB1-Apt) and the carboxyfluorescein FAM-labeled ochratoxin A aptamer (OTA-Apt) were used as two fluorescent probes. The cDNA is anchored to the quenching material gold nanoflowers (AuNFs) by the action of streptavidin (SA) and biotin. Its ends can be complementarily paired with two fluorescent probe bases to form a double-stranded structure. The fluorescence of Cy3 was quenched by AuNFs, and the fluorescence of FAM was quenched by BHQ1 through the fluorescence energy resonance transfer (FRET) effect, forming a fluorescence quenching system. Due to the high affinity of the target and the aptamer, the structure of the aptamer probe changes and detaches from the sensor when AFB1 and OTA are present, resulting in enhanced fluorescence. Under optimal conditions, the linear range of AFB1 was 0.1-100ng/mL (R2 = 0.996), the limit of detection (LOD) was as low as 0.014ng/mL, and the limit of quantification (LOQ) was 0.046ng/mL. The linear range of OTA was 0.1-100ng/mL (R2 = 0.995), the limit of detection (LOD) was as low as 0.027ng/mL, and the limit of quantification (LOQ) was 0.089ng/mL. The sensor had high accuracy in detecting both AFB1 and OTA in real sample analysis. The results of the t test show that there is no significant difference between the results of this study and the high-performance liquid phase (HPLC) method, indicating that the prepared sensor can be used as a potential platform for multiple mycotoxins detection.

Target-responsive aptamer-cross-linked hydrogel sensors for the visual quantitative detection of aflatoxin B1 using exonuclease I-Triggered target cyclic amplification.

For the on-site detection of aflatoxin B1 (AFB1), a DNA hydrogel was prepared as a biosensor substrate, while an AFB1 aptamer was used as the recognition element. An AFB1-responsive aptamer-cross-linked hydrogel sensor was constructed using an enzyme-linked signal amplification strategy; AFB1 binds competitively to the aptamer, causing the hydrogel to undergo cleavage and release horseradish peroxidase (HRP). The addition of exonuclease I (ExoI) to the hydrogel causes the release of AFB1 from the aptamer, promoting additional hydrogel cleavage to release more HRP, ultimately catalysing the reaction between 3,3',5,5'-tetramethylbenzidine and H2O2. The hydrogel sensor exhibited an outstanding sensitivity (limit of detection, 4.93nM; dynamic range, 0-500nM), and its selectivity towards seven other mycotoxins was confirmed. The feasibility and reliability were verified by measuring the AFB1 levels in peanut oil (recoveries, 89.59-95.66%; relative standard deviation, <7%); the obtained results were comparable to those obtained by UPLC-HRMS.