Deoxynivalenol (DON)-Triggered Dual-Color Composite Probe Based on Gold Nanoclusters for Simultaneous Imaging of DON and miR-34a in Living Cells.

Deoxynivalenol (DON) is a mycotoxin secreted by Fusarium species, posing great harm to food safety and human health. Therefore, it is of great significance to study its toxic effects and mechanism. miR-34a is a representative biomarker during the process of DON-induced apoptosis. Herein, a DON-triggered dual-color composite probe was constructed for simultaneous imaging of DON and miR-34a in living cells. The aptamer blocks the recognition sequence of miR-34a to realize DON-triggered cell imaging. The specific binding of DON with its aptamer and HCR induced by miR-34a resulted in the recovery of fluorescence of the dual-color Au NCs. Under the optimal conditions, the correlation between the relative fluorescence intensities of dual-color Au NCs showed good linear relationships with the logarithm of DON and miR-34a concentration, respectively. With the increase in DON concentration (0-20 µg/mL) and stimulation time (0-12 h), the fluorescence of dual-color Au NCs gradually recovered. This dual-color Au NCs composite probe can realize simultaneous detection of DON and miR-34a induced by DON, which is significant for verifying the cytotoxic mechanism of DON.

Hydrogel-integrated sensors for food safety and quality monitoring: Fabrication strategies and emerging applications.

Food safety is a global issue in public hygiene. The accurate, sensitive, and on-site detection of various food contaminants performs significant implications. However, traditional methods suffer from the time-consuming and professional operation, restricting their on-site application. Hydrogels with the merits of highly porous structure, high biocompatibility, good shape-adaptability, and stimuli-responsiveness offer a promising biomaterial to design sensors for ensuring food safety. This review describes the emerging applications of hydrogel-based sensors in food safety inspection in recent years. In particular, this study elaborates on their fabrication strategies and unique sensing mechanisms depending on whether the hydrogel is stimuli-responsive or not. Stimuli-responsive hydrogels can be integrated with various functional ligands for sensitive and convenient detection via signal amplification and transduction; while non-stimuli-responsive hydrogels are mainly used as solid-state encapsulating carriers for signal probe, nanomaterial, or cell and as conductive media. In addition, their existing challenges, future perspectives, and application prospects are discussed. These practices greatly enrich the application scenarios and improve the detection performance of hydrogel-based sensors in food safety detection.

Deoxynivalenol fluorescence aptasensor based on AuCu bimetallic nanoclusters and MoS2.

Aptamers against deoxynivalenol (DON) were selected through capture-systematic evolution of ligands by exponential enrichment. Through isothermal titration calorimetry and fluorimetric assay, aptamer candidate DN-2 demonstrated good affinity to DON with Kd value of 40.36 ± 6.32 nM. Accordingly, a Forster resonance energy transfer aptasensor was fabricated by using the aptamer DN-2 combined with AuCu bimetallic nanoclusters as energy donor and MoS2 nanosheets as energy acceptor. Under the optimal conditions, the fluorescence response was utilized for DON quantitative determination ranging from 5 to 100 ng/mL with a detection limit of 1.87 ng/mL. The practical application of this method was verified in maize flour samples and demonstrated a satisfied recovery of 94.6 ~ 103.1%. The obtained aptamers and their application in DON determination provide a new tool for DON monitoring in various foodstuff.

ZIF-8 base-aptamer "gate-lock" probes enable the visualization of a cascade response between deoxynivalenol and cytochrome c inside living cells.

Zeolitic imidazolate framework (ZIF-8) base-aptamer "gate-lock" biomaterial probes have been synthesized for monitoring intracellular deoxynivalenol (DON) and cytochrome c (cyt c) levels. The aptamer and organic fluorescent dye were regarded as a recognition element and a sensing element, respectively. In the presence of DON, the aptamers of DON and cyt c were specifically bound with the DON and induced cyt c, leading to the dissociation of aptamers from the porous surface of the probes. The gate was subsequently opened to release methylene blue (MB) and Rhodamine 6G (Rh6G), and their fluorescence (emission of MB at 700 nm and Rh6G at 550 nm) significantly recovered within 6 h. Cell imaging successfully monitored the exposure of DON and the biological process of cyt c discharge triggered by the activation of the DON-induced apoptosis pathway. In addition, the response between DON and cyt c was observed during the apoptosis process, which is of high significance for the comprehensive and systematic development of mycotoxins cytotoxicity.

Screening and application of a broad-spectrum aptamer for acyclic guanosine analogues.

Acyclic guanosine analogues, a class of widely used antiviral drugs, can cause chronic toxicity and virus resistance. Therefore, it is essential to establish rapid and accurate methods to detect acyclic guanosine analogues. In this study, five acyclic guanosine analogues (acyclovir, famciclovir, ganciclovir, penciclovir, and valaciclovir) were used as positive targets to obtain broad-spectrum aptamers through Capture-SELEX technology. Real-time quantitative PCR (Q-PCR) was used to monitor the aptamer SELEX process. After the sixteen rounds of selection against mixed targets, sequences were obtained by high-throughput sequencing (HTS). Furthermore, a broad-spectrum aptamer, named CIV6, was found as the higher performance aptamer that was suitable for five acyclic guanosine analogues by graphene oxide (GO) polarization and fluorescence assay. Finally, the aptamer CIV6 was used to construct GO fluorescence assay to detect five acyclic guanosine analogues. The limits of detection (LOD) of acyclovir, famciclovir, ganciclovir, penciclovir, and valaciclovir were 0.48 ng·mL-1, 0.53 ng·mL-1, 0.50 ng·mL-1, 0.56 ng·mL-1, and 0.38 ng·mL-1, respectively.

A colorimetric aptamer-based method for detection of cadmium using the enhanced peroxidase-like activity of Au-MoS2 nanocomposites.

In this work, a colorimetric aptamer-based method for detection of cadmium using gold nanoparticles modified MoS2 nanocomposites as enzyme mimic is established. In short, biotinylated Cd2+ aptamers are immobilized by biotin-avidin binding on the bottoms of the microplate, the complementary strands of Cd2+ aptamers are connected to the Au-MoS2 nanocomposites which have the function of enhanced peroxidase-like activity. The csDNA-Au-MoS2 signal probe and target Cd2+ compete for binding Cd2+ aptamer, the color change can be observed by addition of chromogenic substrate, thereby realizing visual detection of Cd2+. The absorbance of the solution at 450 nm has a clear linear relationship with the Cd2+ concentration. The linear range is 1-500 ng/mL, and the limit of detection is 0.7 ng/mL. The assay was used to test white wine samples, the results are consistent with those of atomic absorption spectrometry; which prove that this method can be used for detection of Cd2+ in real samples.

Fluorometric determination of acetamiprid using molecularly imprinted upconversion nanoparticles.

This paper describes the fabrication of an imprinted fluorescent nanoprobe based on SiO2-coated NaYF4: Yb, Er upconversion nanoparticles (UCNP) encapsulated with a molecularly imprinted polymer (MIP) for determination of acetamiprid. The fluorescent MIP nanoprobe was prepared using UCNP as the material for fluorescence signal readout, acetamiprid as template molecule, methylacrylic acid (MAA) as functional monomer, and ethyleneglycol dimethacrylate (EGDMA) as cross-linking agent. The molecular imprinting layers were immobilized on the surface of the UCNP@SiO2 by polymerization which occurred between the double bonds. UCNP@MIP shows a high selectivity towards acetamiprid with an imprinting factor (IF) of 7.84. When UCNP@MIP combines with acetamiprid, the fluorescence of the UCNP@MIP can be quenched due to the photo-induced electron transfer. Under optimum conditions, the fluorescence method shows a good linear relationship between the decreased fluorescence intensity (with excitation/emission peaks at 980/542 nm) and the variation of acetamiprid in the concentration range 20 to 800 ng mL-1. The limit of detection (LOD) is 8.3 ng mL-1. This fluorescence method was also successfully applied to detect acetamiprid in apple and strawberry samples. The recoveries range from 89.6 to 97.9%, with relative standard deviations between 1.6 and 2.9% (n = 5). Graphical abstractA simple fluorescence nanoprobe which integrates upconversion nanoparticles (UCNPs) and molecular imprinting polymer (MIP) was developed for the determination of acetamiprid. The limit of the detection was determined as 8.3 ng mL-1. The selectivity was enhanced by molecular imprinting, and the sensitivity was improved by the high sensitivity of the fluorescence emitted from the UCNPs.

GO-amplified fluorescence polarization assay for high-sensitivity detection of aflatoxin B1 with low dosage aptamer probe.

Aflatoxin B1 (AFB1) is the most toxic mycotoxin of the aflatoxins (AFs) and shows carcinogenic, teratogenic and mutagenic effects in humans and animals. AFB1 is widely seen in cereal products such as rice and wheat. This research proposed a low-cost, high-sensitivity fluorescence polarization (FP) assay for detection of AFB1 using aptamer biosensors based on graphene oxide (GO). The aptamers labelled with fluorescein amidite (FAM) were adsorbed on the surface of GO through π-π stacking and electrostatic interaction, thus forming aptamer/GO macromolecular complexes. Under these conditions, the local rotation of fluorophores was limited and the system had a high FP value. When there was AFB1 in the system, aptamers were dissociated from the GO surface and combined with AFB1 owing to their specificity to form aptamer/AFB1 complexes. As a result, large changes were observed in the molecular weights of aptamers before, and after, the combination, therefore leading to the apparent changes in FP value. The results showed that when only 10 nM of aptamer was used, the changes in FP and the AFB1 concentration had a favourable linear relationship within 0.05 to 5 nM of AFB1, and the lowest detection limit (LOD) was 0.05 nM. In addition, the recoveries of rice sample extract ranged from 89.2% to 112%. The method is simple, highly sensitive, cost-efficient and shows potential application prospects.

Simultaneous detection of fumonisin B1 and ochratoxin A using dual-color, time-resolved luminescent nanoparticles (NaYF4: Ce, Tb and NH2-Eu/DPA@SiO2) as labels.

A rapid and highly sensitive time-resolved fluorescence (TRF)-based aptasensor for simultaneous recognition of mycotoxins ochratoxin A (OTA) and fumonisin B1 (FB1) was developed using multi-color, Ln3+-doped time-resolved fluorescence nanoparticles (TRF-NPs) (NaYF4: Ce, Tb and NH2-Eu/DPA@SiO2 NPs) coupled with complementary strand DNA (cDNA) as luminescence probe and aptamers-conjugated amine-functionalized Fe3O4 magnetic nanoparticles (MNPs) act as a capture probe. Under the optimized conditions, the time-resolved fluorescence intensities at 544 and 618 nm corresponded with Tb3+ and Eu3+, respectively, were used to measure FB1 (Y = 19,177.1 + (- 12,054.4)x, R2 = 0.9917) and OTA (Y = 4138.8 + (- 11,182.6)x, R2 = 0.9924), respectively. The limits of detection (LODs) for FB1 and OTA were 0.019 pg mL-1 and 0.015 pg mL-1, respectively, which were much lower than previously described methods for simultaneous recognition of mycotoxins OTA and FB1 while detection range varied from 0.0001-0.5 ng mL-1. This aptasensor was effectively applied to quantity FB1 and OTA in maize samples and results were compared with ELISA method. This is the first reported time-resolved fluorescence (TRF)-based aptasensor to detect two agriculturally important toxins in the maize. The developed aptasensor has potential to be used for detection of toxins in food safety fields. Graphical abstract.

Fluorometric determination of lipopolysaccharides via changes of the graphene oxide-enhanced fluorescence polarization caused by truncated aptamers.

A broad-spectrum ssDNA aptamer containing 80 nucleotides (LA80) and capable of binding to four different sources of lipopolysaccharides (LPSs) was truncated. Two strategies are used to produce truncated aptamers of different length. The results show that LA27, a 27-nt aptamer, retained broad-spectrum capability and has a higher affinity (Kd = 46.2 ± 9.5 nM). A graphene oxide based fluorescence polarization assay (excitation/emission wavelengths: 485/520 nm) was worked out using FAM-labeled LA27. It can detect LPSs from Salmonella entericaserotype typhimurium, Pseudomonas aeruginosa 10 and Escherichia coli 055:B5 with enhanced performance (4.8 to 29-fold improvements) compared to LA80. The assay can be performed within 30 min, and the detection limits are 38.7, 88.0 and 154 ng·mL-1, respectively. Graphical abstract Schematic presentation of the assay: A shorter aptamer, with higher affinity than its original aptamer, was obtained by truncated strategies. This core aptamer lead to release easily and enhance the sensivity of the GO-based fluorescence polarization assay.

Surface-enhanced Raman spectroscopic single step detection of Vibrio parahaemolyticus using gold coated polydimethylsiloxane as the active substrate and aptamer modified gold nanoparticles.

A method is described for single-step detection of V. parahaemolyticus in seafood via aptamer-based SERS. A gold-coated polydimethylsiloxane (PDMS) film was used for the enhancement of Raman scattering. The Raman reporter 4-mercaptobenzoic acid was linked to aptamer modified gold nanoparticles (AuNPs) served as a signalling probe. The negatively charged signalling probe was assembled onto the cysteamine-modified Au-PDMS film through electrostatic adsorption. On addition of V. parahaemolyticus, it will be bound by the aptamer as a biorecognition element, and this leads to the dissociation of the signalling probe from the Au-PDMS film. Hence, the Raman signal (at 1592 cm-1) decreases. The assay has a wide linear response that covers the 1.2 × 102 to 1.2 × 106 cfu·mL-1 V. parahaemolyticus concentration range. The detection limit is 12 cfu·mL-1. The method was successfully applied to the determination of V. parahaemolyticus in oyster and salmon samples. Graphical abstract Schematic presentation of a surface-enhanced Raman spectroscopic method for single step detection of Vibrio parahaemolyticus using gold coated polydimethylsiloxane as the active substrate and aptamer modified gold nanoparticles. This solid substrate simplified the analysis procedures and enhanced the sensitivity.

Selection and characterization, application of a DNA aptamer targeted to Streptococcus pyogenes in cooked chicken.

An aptamer against Streptococcus pyogenes was selected and identified, and a fluorescent method based on the reported aptamer was established to detect S. pyogenes in the cooked chicken. Through a twelve rounds of whole-bacterium SELEX (systematic evolution of ligands by exponential enrichment) selection in vitro, a set of aptamers binding to the whole cell of S. pyogenes were generated, harvesting a low-level dissociation constant (Kd) value of 44 ±â€¯5 nmol L-1 of aptamer S-12. Aptamer-based quantification of S. pyogenes in the cooked chicken sample was implemented in a fluorescence resonance energy transfer-based assay by using graphene oxide, resulting in a limit of detection of 70 cfu mL-1. The selected aptamer showed affinity and selectivity recognizing S. pyogenes; besides, more biosensors based on the selected aptamer as a molecular recognition element could be developed in the innovative determinations of S. pyogenes.

Fluorometric determination of Vibrio parahaemolyticus using an F0F1-ATPase-based aptamer and labeled chromatophores.

An F0F1-ATPase-based aptasensor is described for the fluorometric determination of Vibrio parahaemolyticus. Chromatophores containing F0F1-ATPases were first prepared from Rhodospirillum rubrum cells. Then, an aptamer-functionalized chromatophore acts as the capture probe, and a chromatophore labeled with the pH probe fluorescein acts as the signalling probe. In the presence of V. parahaemolyticus, the rotation rate of F0F1-ATPase is decreased due to the formation of the aptamer-chromatophore complex. This leads to a retarded proton flux out of the chromatophores. As a result, the pH value inside the chromatophores is reduced, and the fluorescence of the pH probe F1300 is accordingly decreased. The relative fluorescence varies linearly over the 15 to 1.5 × 106 cfu·mL-1 Vibrio parahaemolyticus concentration range, and the limit of detection is 15 cfu·mL-1. The method was applied to analyze artificially contaminated salmon samples where it showed excellent perfomance. Graphical abstract In this assay, aptamer functionalized chromatophores act as a capture probe, and the fluoresce in labeled chromatophores as signalling probe. The formation of aptamer-chromatophore complex leads to a retarded proton flux out of the chromatophores. As a result, the pH value inside the chromatophores is reduced, and fluorescence intensity is accordingly decreased.

A test strip for ochratoxin A based on the use of aptamer-modified fluorescence upconversion nanoparticles.

An aptamer-based test strip is described for visual and instrumental determination of the mycotoxin ochratoxin A (OTA). It is based on the use of NaYF4:Yb,Er upconversion nanoparticles (UCNPs) as a label for the aptamer and on the competition between OTA and its complementary sequence for an OTA-specific aptamer. To improve the analytical performance, the optical properties of the UCNPs, the fluidity of the UCNP-aptamer conjugate, and the migration rate on the nitrocellulose membranes were investigated. Under the optimal experimental conditions and by using a 980-nm laser, the relative fluorescence intensity (test line value/control line value) is proportional to the logarithm of the OTA concentration over a range from 5 to 100 ng·mL-1 (R2 = 0.9955). The limit of the detection is 1.86 ng·mL-1. This aptamer based flow assay can be performed within 15 min and has no serious cross-sensitivity to potentially interfering species. It was successfully applied to the determination of OTA in spiked wheat and beer samples. Graphical abstract An aptamer-based upconversion fluorescent strip based on the use of NaYF4:Yb,Er nanoparticles was developed for sensitive detection of Ochratoxin A. The limit of the detection was determined as 1.86 ng·mL-1. The assay can be performed within 15 min, indicating its great potential in point-of-care testing.

Screening and development of DNA aptamers as capture probes for colorimetric detection of patulin.

Patulin (PAT) is a kind of mycotoxin that has serious harmful impacts on both food quality and human health. A high-affinity ssDNA aptamer that specifically binds to patulin was generated using systemic evolution of ligands by exponential enrichment (SELEX) assisted by graphene oxide (GO). After 15 rounds of positive and negative selection, a highly enriched ssDNA pool was sequenced and the representative sequences were subjected to binding assays to evaluate their affinity and specificity. Of the eight aptamer candidates tested, the sequence PAT-11 bound to patulin with high affinity and excellent selectivity with a dissociation constant (Kd) of 21.83 ± 5.022 nM. The selected aptamer, PAT-11, was subsequently used as a recognition element to develop a detection method for patulin based on an enzyme-chromogenic substrate system. The colorimetric aptasensor exhibited a linear range from 50 to 2500 pg mL(-1), and the limit of detection was found to be 48 pg mL(-1). The results indicated that GO-SELEX technology was appropriate for the screening of aptamers against small-molecule toxins, offering a promising application for aptamer-based biosensors.

Advances in aptasensors for the detection of food contaminants.

Food safety is a global health objective, and foodborne diseases represent a major crisis in health. Techniques that are simple and suitable for fast screening to detect and identify pathogenic factors in the food chain are vital to ensure food safety. At present, a variety of analytical methods have been reported for the detection of pathogenic agents. Whereas the sensitivity of detection and quantification are still important challenges, we expect major advances from new assay formats and synthetic bio-recognition elements, such as aptamers. Owing to the specific folding capability of aptamers in the presence of an analyte, aptasensors have substantially and successfully been exploited for the detection of a wide range of small and large molecules (e.g., toxins, antibiotics, heavy metals, bacteria, viruses) at very low concentrations. Here, we review the use of aptasensors for the development of highly sensitive and affordable detection tools for food analysis.

Mn(2+)-doped NaYF4:Yb/Er upconversion nanoparticle-based electrochemiluminescent aptasensor for bisphenol A.

A novel aptasensor labeled with Mn(2+)-doped NaYF4:Yb/Er upconversion nanoparticles (NaYF4:Yb,Er/Mn UCNPs) was employed in electrogenerated chemiluminescence (ECL) for the sensitive detection of bisphenol A (BPA). The ECL aptasensor was assembled by immobilizing the thiolated aptamers of BPA covalently on a gold nanoparticle (AuNPs)-modified electrode and pairing with complementary DNA labeled with NaYF4:Yb,Er/Mn UCNPs. The ECL aptasensor can not only rapidly and accurately detect BPA concentrations from 0.05 to 100 ng/mL with a detection limit of 0.037 ng/mL but also provides a new platform for ECL applications based on the use of upconversion nanoparticles as a promising alternative material. Graphical Abstract The NaYF4:Yb,Er/Mn UCNPs combining with the BPA aptamer serving as recognition elements create a ECL platform for the sensitive detection of bisphenol A. The change in ECL signals induced by aptamer-target interactions was measured and a significant decrease in intensity was found on interaction with BPA in the concentration range of 0.05 to 100 ng/mL.

Upconversion luminescence resonance energy transfer-based aptasensor for the sensitive detection of oxytetracycline.

In this work, a biosensor based on luminescence resonance energy transfer (LRET) from NaYF4:Yb,Tm upconversion nanoparticles (UCNPs) to SYBR Green I has been developed. The aptamers are covalently linked to UCNPs and hybridized with their complementary strands. The subsequent addition of SYBR Green allows SYBR Green I to insert into the formed double-stranded DNA (dsDNA) duplex and brings the energy donor and acceptor into close proximity, leading to the fluorescence of UCNPs transferred to SYBR Green I. When excited at 980 nm, the UCNPs emit luminescence at 477 nm, and this energy is transferred to SYBR Green I, which emits luminescence at 530 nm. In the presence of oxytetracycline (OTC), the aptamers prefer to bind to its corresponding analyte and dehybridize with the complementary DNA. This dehybridization leads to the liberation of SYBR Green I, which distances SYBR Green I from the UCNPs and recovers the UCNPs' luminescence. Under optimal conditions, a linear calibration is obtained between the ratio of I530 to I477 nm (I530/I477) and the OTC concentration, which ranges from 0.1 to 10 ng/ml with a limit of detection (LOD) of 0.054 ng/ml.

Chemiluminescent aptasensor for chloramphenicol based on N-(4-aminobutyl)-N-ethylisoluminol-functionalized flower-like gold nanostructures and magnetic nanoparticles.

A novel chemiluminescent aptasensor for the highly sensitive detection of chloramphenicol (CAP) in milk was successfully developed using biotinylated CAP aptamer-functionalized magnetic nanoparticles (MNPs) as capture probes and thiolated hybridized complementary strand-modified N-(4-aminobutyl)-N-ethylisoluminol (ABEI)-functionalized flower-like gold nanostructures (AuNFs) as signal probes. P-iodophenol (PIP) was also added to form an ABEI-H2O2-PIP steady-state chemiluminescence (CL) system. Based on a competitive format, the CL intensity was negatively correlated with the concentration of CAP in the range of 0.01-0.20 ng/mL and the detection limit was 0.01 ng/mL in buffer and 1 ng/mL in milk. The proposed method was successfully applied to measure CAP in milk samples and compared to a commercial ELISA method. The high sensitivity of AuNFs, excellent selectivity and stability of aptamers, and good overall stability of the chemiluminescent bioassay with magnetic separation make them a promising approach for the detection of small molecular illegal additives. Additionally, the high sensitivity, easy operation, and good reproducibility exhibited by the stable chemiluminescent bioassay demonstrate its applicability for the trace detection of CAP in applications, such as animal husbandry.

Simultaneous detection of microcysin-LR and okadaic acid using a dual fluorescence resonance energy transfer aptasensor.

Algal toxins can cause neurovirulence, hepatotoxicity, and cytotoxicity in humans through the consumption of contaminated water and food. In this work, we presented a novel aptasensor for the simultaneous detection of two algal toxins, microcysin-LR (MC-LR) and okadaic acid (OA). This system employed green and red upconversion nanoparticle (UCNP) luminescence as the donors and two quenchers (BHQ1 and BHQ3) as the corresponding acceptors. The two donor-acceptor couples were fabricated by hybridizing the aptamers with their corresponding complementary DNA. The results indicated that the green and red upconversion luminescence could be quenched by the quencher probes because of their highly overlapping spectrum. In the presence of MC-LR and OA, the aptamers preferred to bind to their corresponding analytes and de-hybridize with the complementary DNA. This effect became sufficiently large to prevent green and red luminescence quenching. Under the optimized experimental conditions, the relative luminescence intensity increased as the algal toxin concentrations increased, allowing for the quantification of MC-LR and OA. The relationships between the luminescence intensity and plotting logarithms of algal toxin concentrations were linear in the range from 0.1 to 50 ng mL(-1) for MC-LR and OA. As a practical application, this type of dual fluorescence resonance energy transfer (FRET) aptasensor was used to monitor the MC-LR and OA levels in naturally contaminated food samples such as fish and shrimps.