Aptamer-Based Fluorescent Determination of Salmonella paratyphi A Using Phi29-DNA Polymerase-Assisted Cyclic Amplification

A rapid and ultrasensitive fluorescence aptasensor was developed for the detection of Salmonella paratyphi A based on aptamer and Phi29-DNA polymerase-assisted cyclic signal amplification. The method employed a designed arched probe, consisting of an aptamer and a primer, with a designed hairpin probe. The quenching groups and fluorescent groups were modified at the 3′ and 5′ ends of the hairpin probe, respectively. In the absence of the target, the primer was not released and the hairpin probe was not opened to produce fluorescence. The addition of target led to the release of the primer, which hybridized with the hairpin probe and triggered the chain-displacement polymerase reaction and produced a high fluorescence intensity. Under the optimized conditions, the linear range of this aptasensor was from 10² CFU·mL^?1 to 10⁸ CFU·mL^?1 with a detection limit of 10² CFU·mL^?1. Compared with other reported fluorescence detection methods, this approach has two advantages. First, this fluorescence aptasensor does not require nanomaterials as the quencher, which reduces the cost and saves time. Second, the chain-displacement polymerase reaction was used in this fluorescence aptasensor to amplify the signals, which further enhanced the sensitivity and lowered the detection limit. As this method was suitable for the detection of Salmonella paratyphi A in milk samples and potentially other bacteria, environmental monitoring and related food safety analysis should also be possible by this approach.     

基于酶辅助靶标循环的适体传感器灵敏检测中药中黄曲霉毒素B1

该文基于酶辅助靶标循环信号放大策略构建了用于黄曲霉毒素B1(AFB1)高灵敏检测的化学发光适体传感器。以G-四链体/氯化血红素DNA酶为信号分子设计了免标记的适体探针H1-S1和发夹探针H2。适体探针结合目标AFB1,在核酸外切酶I辅助下,触发靶标循环反应产生发夹H1。发夹H1与H2杂交,释放出完整的G-四链体序列,并进一步与氯化血红素结合形成G-四链体/氯化血红素DNA酶。DNA酶通过催化氧化鲁米诺-H2O2化学发光体系产生化学发光信号,实现AFB1的放大检测。在最优实验条件下,化学发光强度与AFB1质量浓度的对数在0.001~100 ng/mL范围内呈良好的线性关系,相关系数(r2)为0.9955,检出限为0.93 pg/mL,回收率为93.7%~107%。该适体传感器操作简单、灵敏度高、特异性好,在黄曲霉毒素污染检测方面具有良好的应用前景。     

A novel electrochemiluminescence aptasensor for protein based on a sensitive N-(aminobutyl)-N-ethylisoluminol-functionalized gold nanoprobe

A novel electrochemiluminescence (ECL) aptasensor for platelet-derived growth factor B chain (PDGF-BB) assay was developed by assembling N-(aminobutyl)-N-ethylisoluminol functionalized gold nanoparticles (ABEI-AuNPs) with aptamers as nanoprobes. In the protocol, the biotinylated aptamer capture probes were first immobilized on a streptavidin coated gold nanoparticle (AuNPs) modified electrode, afterwards, the target PDGF-BB and the ABEI-AuNPs tagged aptamer signal probe were successively attached to the modified electrode by virtue of the dimer structure of PDGF-BB to fabricate a "sandwich" conjugate modified electrode, i.e. an aptasensor. ECL measurement was carried out with a double-step potential in carbonate buffer solution containing H(2)O(2). The aptasensor showed high sensitivity and selectivity toward PDGF-BB and specificity toward PDGF-BB aptamer. The detection limit was as low as 2.7 × 10(-14) M. In this work, the ABEI-AuNPs synthesized by a simple seed growth method have been successfully used as aptamer labels, which greatly amplified the ECL signal by binding numbers of ABEI molecules on the surface of AuNPs. The ABEI-AuNPs signal amplification is superior to other reported signal amplification strategies based on aptamer-related polymerase chain reaction or functionalized nanoparticles in simplicity, stability, labeling property and practical applicability. And the ABEI-AuNPs based nanoprobe is more sensitive than the luminol functionalized AuNPs based nanoprobe. Moreover, such an ultra-sensitive and low-cost assay can be accomplished with a simple and fast procedure by using a simple ECL instrumentation. The aptasensor was also applied for the detection of PDGF-BB in human serum samples, showing great application potential. Given these advantages, the ECL aptasensor is well suited for the direct, sensitive and rapid detection of protein in complex clinical samples.     

Sandwich-type aptasensor employing modified aptamers and enzyme-DNA binding protein conjugates

The use of aptamers in various analytical applications as molecular recognition elements and alternative to antibodies has led to the development of various platforms that facilitate the sensitive and specific detection of targets ranging from small molecules and proteins to whole cells. The goal of this work was to design a universal and adaptable sandwich-type aptasensor exploiting the unique properties of DNA binding proteins. Specifically, two different enzyme-DNA binding protein conjugates, GOx-dHP and HRP-scCro, were used for the direct detection of a protein using two aptamers for target capture and detection. The specific dsDNA binding sequence for each DNA binding protein tag was incorporated in the form of a hairpin at one end of each aptamer sequence during the synthesis step. Detection was accomplished by an enzymatic (GOx/HRP) cascade reaction after the binding of each enzyme conjugate to its corresponding binding sequence on each aptamer. The proposed sandwich-type aptasensor was validated for the detection of thrombin, which is one of the most commonly used model targets with known dual aptamers. The limit of detection accomplished was 0.92 nM which is comparable with other colorimetric platforms reported in the literature. The sensitivity of the aptasensor was easily modulated by changing the number of dsDNA binding sites incorporated in the aptamer sequences, thus controlling the enzyme stoichiometry. Finally, the potential use of the proposed sensing approach for real sample testing was demonstrated using spiked human plasma and no significant matrix effects were observed when up to 2% plasma was used.

     

An Electrochemical Aptasensor for Detection of Thrombin based on Target Protein‐induced Strand Displacement

A sensitive electrochemical aptasensor for detection of thrombin based on target protein‐induced strand displacement is presented. For this proposed aptasensor, dsDNA which was prepared by the hybridization reaction of the immobilized probe ssDNA (IP) containing thiol group and thrombin aptamer base sequence was initially immobilized on the Au electrode by self‐assembling via Au? S bind, and a single DNA labeled with CdS nanoparticles (DP‐CdS) was used as a detection probe. When the so prepared dsDNA modified Au electrode was immersed into a solution containing target protein and DP‐CdS, the aptamer in the dsDNA preferred to form G‐quarter structure with the present target protein resulting that the dsDNA sequence released one single strand and returned to IP strand which consequently hybridized with DP‐CdS. After dissolving the captured CdS particles from the electrode, a mercury‐film electrode was used for electrochemical detection of these Cd²⁺ ions which offered sensitive electrochemical signal transduction. The peak current of Cd²⁺ ions had a good linear relationship with the thrombin concentration in the range of 2.3×10^?9–2.3×10^?12 mol/L and the detection limit was 4.3×10^?13 mol/L of thrombin. The detection was also specific for thrombin without being affected by the coexistence of other proteins, such as BSA and lysozyme.     

Dual signal amplification strategy for amperometric aptasensing using hydroxyapatite nanoparticles. Application to the sensitive detection of the cancer biomarker platelet-derived growth factor BB

The authors describe a dual signal amplification strategy for improving the sensitivity of electrochemical aptasensor. Hydroxyapatite nanoparticles (HAP-NPs) serve as the support for deposition of the respective aptamer. Both the HAP-NPs and the aptamer contain phosphate groups which can react with molybdate to form a redox-active molybdophosphate precipitate on the surface of a glassy carbon electrode (GCE). On applying a relatively low voltage of 0.21 V (vs. Ag/AgCl), a current is generated whose intensity depends on the concentration of the analyte. The cancer biomarker platelet-derived growth factor BB (PDGF-BB) is chosen as a model antigen (analyte). The assay works by sequential deposition of antibody against PDGF-BB, analyte (PDGF-BB) and anti-PDGF-BB aptamer modified HAP-NPs on the GCE to form a sandwich structure. The amperometric signal is linear in the 0.1 pg.mL^?1 to 10 ng.mL^?1 PDGF-BB concentration range, with a detection limit as low as 50 fg.mL^?1. The assay was successfully applied to the determination of PDGF-BB in serum samples. In our perception, this signal amplification strategy has a wide scope in that it can be adapted to the preparation of other aptasensors for biomarkers and related species.

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Graphical abstract Schematic of an electrochemical aptasensor based on dual signal amplification strategy. It was applied to the detection of cancer biomarker platelet-derived growth factor BB (PDGF-BB). Hydroxyapatite (HAP) nanoparticles were chosen for the immobilization of aptamers to increase the loading of aptamers.

     

A sensitive electrochemical aptasensor for ATP detection based on exonuclease III-assisted signal amplification strategy

A target-induced structure-switching electrochemical aptasensor for sensitive detection of ATP was successfully constructed which was based on exonuclease III-catalyzed target recycling for signal amplification. With the existence of ATP, methylene blue (MB) labeled hairpin DNA formed G-quadruplex with ATP, which led to conformational changes of the hairpin DNA and created catalytic cleavage sites for exonuclease III (Exo III). Then the structure-switching DNA hybridized with capture DNA which made MB close to electrode surface. Meanwhile, Exo III selectively digested aptamer from its 3′-end, thus G-quadruplex structure was destroyed and ATP was released for target recycling. The Exo III-assisted target recycling amplified electrochemical signal significantly. Fluorescence experiment was performed to confirm the structure-switching process of the hairpin DNA. In fluorescence experiment, AuNPs–aptamer conjugates were synthesized, AuNPs quenched fluorescence of MB, the target-induced structure-switching made Exo III digested aptamer, which restored fluorescence. Under optimized conditions, the proposed aptasensor showed a linear range of 0.1–20 nM with a detection limit of 34 pM. In addition, the proposed aptasensor had good stability and selectivity, offered promising choice for the detection of other small molecules.     

Enzyme-free surface plasmon resonance aptasensor for amplified detection of adenosine via target-triggering strand displacement cycle and Au nanoparticles

Herein, we combine the advantage of aptamer technique with the amplifying effect of an enzyme-free signal-amplification and Au nanoparticles (NPs) to design a sensitive surface plasmon resonance (SPR) aptasensor for detecting small molecules. This detection system consists of aptamer, detection probe (c-DNA1) partially hybridizing to the aptamer strand, Au NPs-linked hairpin DNA (Au-H-DNA1), and thiolated hairpin DNA (H-DNA2) previously immobilized on SPR gold chip. In the absence of target, the H-DNA1 possessing hairpin structure cannot hybridize with H-DNA2 and thereby Au NPs will not be captured on the SPR gold chip surface. Upon addition of target, the detection probe c-DNA1 is forced to dissociate from the c-DNA1/aptamer duplex by the specific recognition of the target to its aptamer. The released c-DNA1 hybridizes with Au-H-DNA1 and opens the hairpin structure, which accelerate the hybridization between Au-H-DNA1 and H-DNA2, leading to the displacement of the c-DNA1 through a branch migration process. The released c-DNA1 then hybridizes with another Au-H-DNA1 probe, and the cycle starts anew, resulting in the continuous immobilization of Au-H-DNA1 probes on the SPR chip, generating a significant change of SPR signal due to the electronic coupling interaction between the localized surface plasma of the Au NPs and the surface plasma wave. With the use of adenosine as a proof-of-principle analyte, this sensing platform can detect adenosine specifically with a detection limit as low as 0.21 pM, providing a simple, sensitive and selective protocol for small target molecules detection.     

An electronic channel switching-based aptasensor for ultrasensitive protein detection

Due to the ubiquity and essential of the proteins in all living organisms, the identification and quantification of disease-specific proteins are particularly important. Because the conformational change of aptamer upon its target or probe/target/probe sandwich often is the primary prerequisite for the design of an electrochemical aptameric assay system, it is extremely difficult to construct the electrochemical aptasensor for protein assay because the corresponding aptamers cannot often meet the requirement. To circumvent the obstacles mentioned, an electronic channel switching-based (ECS) aptasensor for ultrasensitive protein detection is developed. The essential achievement made is that an innovative sensing concept is proposed: the hairpin structure of aptamer is designed to pull electroactive species toward electrode surface and makes the surface-immobilized IgE serve as a barrier that separates enzyme from its substrate. It seemingly ensures that the ECS aptasensor exhibits most excellent assay features, such as, a detection limit of 4.44 × 10⁻⁶ μg mL⁻¹ (22.7 fM, 220 zmol in 10-μL sample) (demonstrating a 5 orders of magnitude improvement in detection sensitivity compared with classical electronic aptasensors) and dynamic response range from 4.44 × 10⁻⁶ to 4.44 × 10⁻¹ μg mL⁻¹. We believe that the described sensing concept here might open a new avenue for the detection of proteins and other biomacromolecules.     

Label-free aptasensor for platelet-derived growth factor (PDGF) protein

A label-free aptasensor for platelet-derived growth factor (PDGF) protein is reported. The aptasensor uses mixed self-assembled monolayers (SAMs) composed of a thiol-modified PDGF binding aptamer and 6-mercaptohexanol (MCH) on a gold electrode. The SAMs were characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV) before and after binding of the protein using [Fe(CN)₆]^3−/4−, a redox marker ion as an indicator for the formation of a protein-aptamer complex. The CVs at the PDGF modified electrode showed significant differences, such as changes in the peak currents and peak-to-peak separation, before and after binding of the target protein. The EIS spectra, in the form of Nyquist plots, were analyzed with a Randles circuit while the electron transfer resistance R_ct was used to monitor the binding of the target protein. The results showed that, without any modification to the aptamer, the target protein can be recognized effectively at the PDGF binding aptamer SAMs at the electrode surface. Control experiments using non-binding oligonucleotides assembled at the electrode surfaces also confirmed the results and showed that there was no formation of an aptamer-protein complex. The DPV signal at the aptamer functionalized electrode showed a linearly decreased marker ion peak current in a protein concentrations range of 1-40 nM. Thus, label-free detection of PDGF protein at an aptamer modified electrode has been demonstrated.     

Ultrasensitive aptamer-based bio bar code immunomagnetic separation and electrochemiluminescence method for the detection of protein

An ultrasensitive aptamer-based bio bar code immunomagnetic separation and electrochemiluminescence (IM-ECL) method for the detection of protein is developed. The target protein is captured by biotin-labeled aptamer (biotin probe) and [Ru(bpy)₃]²⁺ (TBR)-Au bio bar code-labeled aptamer (ECL nanoprobe), to form a double aptamer–protein sandwich complex. The complex is then immobilized on the streptavidin microbeads through biotin–streptavidin linkage and detected by ECL assay. The ECL signal of the target protein is amplified by the TBR-bio bar code DNAs. As an example, platelet-derived growth factor B-chain homodimer (PDGF-BB) was detected by the method. Experimental results show that the detection limit of the assay is 1 pM of PDGF-BB. A calibration curve with a linearity range from 1 pM to 10 nM is established, thus, make quantitative analysis possible. The method has been used to detect PDGF-BB in fetal calf serum with minimum background interference. Due to the wide availability of aptamer for numerous proteins, this aptamer-based bio bar code IM-ECL method holds great promise in protein detection.     

Detection of oncoprotein platelet-derived growth factor using a fluorescent signaling complex of an aptamer and TOTO

There have recently been advances in the application of aptamers, a new class of nucleic acids that bind specifically with target proteins, as protein recognition probes for biomedical study. The development of a signaling aptamer with the capability of simple and rapid real-time detection of disease-related proteins has attracted increasing interest. We have recently reported a new protein-detection strategy using a signaling aptamer based on a DNA molecular light-switching complex, [Ru(phen)₂(dppz)]²⁺. In this work we have used the commercially available DNA-intercalating dye, TOTO, to replace [Ru(phen)₂(dppz)]²⁺ for detection of oncoprotein platelet-derived growth factor BB (PDGF-BB), a potential cancer marker. Taking advantage of the high affinity of the aptamer to PDGF-BB and the sensitive fluorescence change of the aptamer–TOTO signaling complex on protein binding, PDGF-BB was detected in physiological buffer with high selectivity and sensitivity. The detection limit was 0.1 nmol L⁻¹, which was better than that of other reported aptamer-based methods for PDGF-BB, including that using [Ru(phen)₂(dppz)]²⁺. The method is very simple with no need for covalent labeling of the aptamer or probe synthesis. It facilitates wide application of the signaling mechanism to the analysis and study of cancer markers and other proteins.      

A homogeneous hemin/G-quadruplex DNAzyme based turn-on chemiluminescence aptasensor for interferon-gamma detection via in-situ assembly of luminol functionalized gold nanoparticles,deoxyribonucleic acid,interferon-gamma and hemin

A homogeneous hemin/G-quadruplex DNAzyme (HGDNAzyme) based turn-on chemiluminescence aptasensor for interferon-gamma (IFN-γ) detection is developed, via dynamic in-situ assembly of luminol functionalized gold nanoparticles (lum-AuNPs), DNA, IFN-γ and hemin. The G-quadruplex oligomer of the HGDNAzyme was split into two halves, which was connected with the complementary sequence of P1 (IFN-γ-binding aptamer) to form the oligonucleotide P2. P2 hybridized with IFN-γ-binding aptamer and meanwhile assembled onto lum-AuNPs through biotin–streptavidin specific interaction. When IFN-γ was recognized by aptamer, P2 was released into the solution. The two lateral portions of P2 combined with hemin to yield the catalytic hemin/G-quadruplex DNAzyme, which amplified the luminol oxidation for a turn-on chemiluminescence signaling. Based on this strategy, the homogeneous aptasensor enables the facile detection of IFN-γ in a range of 0.5–100 nM. Moreover, the aptasensor showed high sensitivity (0.4 nM) and satisfactory specificity, pointing to great potential applications in clinical analysis.     

Development of Electrochemical Impedance Spectroscopy Based Malaria Aptasensor Using HRP‐II as Target Biomarker

Current demand for a stable, low cost and sensitive malaria sensor has prompted to explore novel recognition systems that can substitute widely used protein based labile biorecognition elements to be used in point of care diagnostic devices. Here, we report a novel ssDNA aptamer of 90 mer sequence developed by SELEX process against HRP‐II, a specific biomarker for Plasmodium falciparum strains. High stability of the secondary structure of the isolated aptamer was discerned from its free energy of folding of −20.40 kcal mole⁻¹. The binding constant (K_d) of the aptamer with HRP‐II analysed by isothermal titration calorimetry was ∼1.32 μM. Circular dichroism studies indicated B form of the aptamer DNA. The aptamer was chemically immobilized on a gold electrode surface through a self‐assembled monolayer of dithio‐bis(succinimidyl) propionate to produce the aptasensor. The step wise modification of the layers over the gold electrode during fabrication of the aptasensor was confirmed by cyclic voltammetry. The aptasensor was then challenged with different concentration of HRP‐II and analysed the interaction signals through electrochemical impedance spectroscopy. The impedance signal behaved reciprocally with the increasing concentrations of the target in the sample from which a dynamic range of 1 pM–500 pM (R²=0.99) and LOD of ∼3.15 pM were discerned. The applicability of the developed aptasensor to detect HRP‐II in mimicked real sample was also validated.     

Label-free aptamer-based chemiluminescence detection of adenosine

We have developed a novel sensitive chemiluminescence (CL) aptasensor for the target assay as exemplified by using adenosine as a model target. In this work, we have demonstrated the signaling mechanism to make detection based on magnetic separation and 3,4,5-trimethoxyl-phenylglyoxal (TMPG), a special CL reagent as the signaling molecule, which reacts instantaneously with guanine nucleobases (G) of adenosine-binding aptamer strands. Briefly, amino-functioned capture DNA sequences are immobilized on the surface of carboxyl-modified magnetic beads, and then hybridized with label-free G-rich (including 15 guanine nucleobases) adenosine-binding aptamer strands to form our CL aptasensor. Upon the introduction of adenosine, the aptamer on the surface of magnetic beads is triggered to make structure switching to the formation of the adenosine/aptamer complex. Consequently, G-rich aptamer strands are forced to dissociate from magnetic beads sensing interface, resulting in a decrease of CL signal. The decrement of peak signal is proportional to the amount of adenosine. The effects of the amounts of capture DNA, aptamer, magnetic beads are investigated and optimized. It was found that the CL intensity had a linear dependency on the concentration of adenosine in the range of 4 × 10⁻⁷ to 1 × 10⁻⁵ M. With a low detection limit of 8 × 10⁻⁸ M and simplicity in CL detection, this novel technique will offer a great promise for future target/aptamer analysis.     

Hybridization chain reaction-based aptameric system for the highly selective and sensitive detection of protein

We introduce here a novel assay for the detection of platelet-derived growth factor BB (PDGF-BB) via hybridization chain reaction (HCR) based on an aptameric system, where stable DNA monomers assemble only upon exposure to a target PDGF-BB aptamer. In this process, two complementary stable species of biotinylated DNA hairpins coexist in solution until the introduction of initiator aptamer strands triggers a cascade of hybridization events that yields nicked double helices analogous to alternating copolymers. In detail, the aptamer firstly opens the hairpins in the solution, creating long concatemers, and then reacts with the antibody captured PDGF-BB on the well surface. Moreover, several experimental conditions including different PDGF-BB aptamers, the spacer length of the selected aptamer and hairpin, etc. are investigated and optimized. Our results show that the coupling of HCR to aptamer triggers for the amplification detection of PDGF-BB achieves a better performance in the fluorescence detection of PDGF-BB as compared to the traditional antibody-antigen-aptamer assays. Upon modification, the approach presented herein could be extended to detect other types of targets. We believe such advancements will represent a significant step towards improved diagnostics and more personalized medical treatment and environmental monitoring.     

Ultrasensitive one-step rapid detection of ochratoxin A by the folding-based electrochemical aptasensor

A one-step electrochemical aptasensor using the thiol- and methylene blue- (MB-) dual-labeled aptamer modified gold electrode for determination of ochratoxin A (OTA) was presented in this research. The aptamer against OTA was covalently immobilized on the surface of the electrode by the self-assembly effect and used as recognition probes for OTA detection by the binding induced folding of the aptamer. Under the optimal conditions, the developed electrochemical aptasensor demonstrated a wide linear range from 0.1 pg mL⁻¹ to 1000 pg mL⁻¹ with the limit of detection (LOD) of 0.095 pg mL⁻¹, which was an extraordinary sensitivity compared with other common methods for OTA detection. Moreover, as a practical application, this proposed electrochemical aptasensor was used to monitor the OTA level in red wine samples without any special pretreatment and with satisfactory results obtained. Study results showed that this electrochemical aptasensor could be a potential useful platform for on-site OTA measurement in real complex samples.     

Aptamer-functionalized Ti3C2-MXene Nanosheets with One-step Potentiometric Detection of Programmed Death-ligand 1

This communication describes a simple sensitive one-step potentiometric aptasensing method for quantitative detection of a referenced therapeutic biomarker (programmed death-ligand 1, PD−L1). The aptasensor is constructed by modifying PD−L1-specific aptamer on Ti₃C₂-MXene nanosheets-functionalized electrode. Introduction of PD−L1 induces the specific reaction between PD−L1 and aptamer, thereby resulting in the change of spatial structures. The surface electric potential of modified electrode is shifted upon addition of PD−L1 proteins before and after the reaction of aptamer with the analyte. Interestingly, potentiometric aptamer with Ti₃C₂-MXene nanosheets can achieve a higher sensitivity and a lower detection limit toward target PD−L1 relative to aptamer-modified electrode. Experimental results indicated that the linear range and detection limit of using Ti₃C₂-MXene nanosheets were 0.01–100 ng mL⁻¹ and 7.8 pg mL⁻¹ PD−L1, respectively. Meanwhile, the specificity, reproducibility, storing stability and accuracy of potentiometric aptasensor are acceptable for the screening of PD−L1 in human serum samples.     

A novel fluorescent aptasensor based on mesoporous silica nanoparticles for the selective detection of sulfadiazine in edible tissue

Sulfadiazine (SDZ) is a broad-spectrum antibiotic used to treat bacterial infections in animals, and SDZ residues in food can be harmful to human health. As a result, an aptasensor based on silica nanoparticles was developed for the rapid detection of SDZ. An aptamer that specifically binds to SDZ was obtained using graphene oxide-SELEX and further truncated to a 13 nt sequence (SDZ30-1:5′-AACCCAATGGGAT-3′), which has a high affinity (K_d = 65.72 nM). In addition, it was found by molecular simulation that a steric hindrance could prevent the target molecule from entering the binding pocket formed by the key base “TGG”, which affects the total binding free energy of SDZ30-1 and the target molecule, thereby affecting the affinity of SDZ30-1 to the target. The SDZ30-1 was selected as the fluorescent probe to establish an aptasensor for the detection of SDZ residues in milk and honey. The aptasensor exhibited a wide dynamic linear range (3.125 – 100 ng/mL) and a limit of detection (LOD = 1.68 ng/mL). The aptasensor in spiked samples recovered at a rate of 95.12 – 105.47%, with a coefficient of variation of less than 13.18 %. The results of aptasensor were positively correlated with those of HPLC (R² > 0.8687). Based on the above results, it could be inferred that the aptasensor can be used sensitively and rapidly for the detection of SDZ residues in edible tissue.     

A highly sensitive fluorescence resonance energy transfer aptasensor for staphylococcal enterotoxin B detection based on exonuclease-catalyzed target recycling strategy

An ultrasensitive fluorescence resonance energy transfer (FRET) bioassay was developed to detect staphylococcal enterotoxin B (SEB), a low molecular exotoxin, using an aptamer-affinity method coupled with upconversion nanoparticles (UCNPs)-sensing, and the fluorescence intensity was prominently enhanced using an exonuclease-catalyzed target recycling strategy. To construct this aptasensor, both fluorescence donor probes (complementary DNA₁–UCNPs) and fluorescence quencher probes (complementary DNA₂–Black Hole Quencher₃ (BHQ₃)) were hybridized to an SEB aptamer, and double-strand oligonucleotides were fabricated, which quenched the fluorescence of the UCNPs via FRET. The formation of an aptamer–SEB complex in the presence of the SEB analyte resulted in not only the dissociation of aptamer from the double-strand DNA but also both the disruption of the FRET system and the restoration of the UCNPs fluorescence. In addition, the SEB was liberated from the aptamer–SEB complex using exonuclease I, an exonuclease specific to single-stranded DNA, for analyte recycling by selectively digesting a particular DNA (SEB aptamer). Based on this exonuclease-catalyzed target recycling strategy, an amplified fluorescence intensity could be produced using different SEB concentrations. Using optimized experimental conditions produced an ultrasensitive aptasensor for the detection of SEB, with a wide linear range of 0.001–1 ng mL⁻¹ and a lower detection limit (LOD) of 0.3 pg mL⁻¹ SEB (at 3σ). The fabricated aptasensor was used to measure SEB in a real milk samples and validated using the ELISA method. Furthermore, a novel aptasensor FRET assay was established for the first time using 30 mol% Mn²⁺ ions doped NaYF₄:Yb/Er (20/2 mol%) UCNPs as the donor probes, which suggests that UCNPs are superior fluorescence labeling materials for food safety analysis.