Assembly of a multivalent aptamer for efficient inhibition of thermostable direct hemolysin toxicity induced by Vibrio parahaemolyticus.

Thermostable direct hemolysin (TDH) is a key virulence factor of Vibrio parahaemolyticus, capable of causing seafood-mediated outbreaks of gastroenteritis, posing a threat to the aquatic environment and global public health. In the present study, we explored a multivalent aptamer-mediated inhibition strategy to mitigate TDH toxicity. Based on the characteristic structure of TDH, a stable multivalent aptamer, Ap3-5, was rationally designed by truncation, key fragment evolution, and end fixation. Ap3-5 exhibited strong affinity (Kd=39.24 nM), and thermal (Tm=57.6 °C) and enzymatic stability. In silico studies also revealed that Ap3-5 occupied more active sites of TDH and covered its central pore, indicating its potential as a blocking agent for inhibiting TDH toxicity. In the hemolysis assay, Ap3-5 significantly suppressed the hemolytic effect of TDH. A cellular study revealed a substantial (∼80 %) reduction in TDH cytotoxicity. Supporting these findings, in vivo trials confirmed the inhibitory action of Ap3-5 on both the acute and intestinal toxicity of TDH. Overall, benefiting from the strong binding affinity, high stability, and multisite occupation of the multivalent aptamer with TDH, Ap3-5 displayed robust potential against TDH toxicity by inhibiting membrane pore formation, providing a new approach for alleviating bacterial infections.

Recent advances in the construction strategy, functional properties, and biosensing application of self-assembled triangular unit-based DNA nanostructures.

Research on self-assembled deoxyribonucleic acid (DNA) nanostructures with different shapes, sizes, and functions has recently made rapid progress owing to its biocompatibility, programmability, and stability. Among these, triangular unit-based DNA nanostructures, which are typically multi-arm DNA tiles, have been widely applied because of their unique structural rigidity, spatial flexibility, and cell permeability. Triangular unit-based DNA nanostructures are folded from multiple single-stranded DNA using the principle of complementary base pairing. Its shape and size can be determined using pre-set scaffold strands, segmented base complementary regions, and sequence lengths. The resulting DNA nanostructures retain the desired sequence length to serve as binding sites for other molecules and obtain satisfactory results in molecular recognition, spatial orientation, and target acquisition. Therefore, extensive research on triangular unit-based DNA nanostructures has shown that they can be used as powerful tools in the biosensing field to improve specificity, sensitivity, and accuracy. Over the past few decades, various design strategies and assembly techniques have been established to improve the stability, complexity, functionality, and practical applications of triangular unit-based DNA nanostructures in biosensing. In this review, we introduce the structural design strategies and principles of typical triangular unit-based DNA nanostructures, including triangular, tetrahedral, star, and net-shaped DNA. We then summarize the functional properties of triangular unit-based DNA nanostructures and their applications in biosensing. Finally, we critically discuss the existing challenges and future trends.

Discovery and design of an aptamer that inhibits Shiga toxin type 2 activity by blocking Stx2 B subunit-Gb3 interaction.

Shiga toxin (Stx) is the definitive virulence factor of Stx-producing Escherichia coli. This bacterial pathogen can contaminate food and threaten human health. Binding of the B subunit of Stx to the specific receptor globotriaosylceramide (Gb3) on the cell membrane is a key step for Stx to enter cells and exert its toxicity. In this work, we aimed to screen for aptamers targeting the Stx 2 B subunit, to interfere with the interaction of Stx2 B subunit and Gb3, thereby blocking Stx2 from entering cells. The results of molecular simulation docking, competitive ELISA, flow cytometry, and laser confocal microscopy confirmed that aptamers S4, S5, and S6 can mediate the interaction between Stx2 B subunit and Gb3. To further improve the inhibition effect, multiple aptamer sequences were tailored and were fused. The bivalent modification aptamer B2 inhibited Stx2 toxicity to Vero cells with inhibition rate of 53 %. Furthermore, the aptamer B2 reduced Stx2 damage to the mice, indicating that it has great potential to interfere with Stx2 binding to Gb3 receptors in vivo and in vitro. This work provides a theoretical and experimental basis for the application of aptamers in the inhibition of Stx2 toxicity and control of food hazards.

Detection and simultaneous imaging of acrylamide, miR-21 and miR-221 based on multicolor aggregation-induced emission nanoparticles and DNAzyme walker.

Acrylamide (AA) in heat-processed foods has emerged as a global health problem, mainly carcinogenic, neurotoxic, and reproductive toxicity, and an increasing number of researchers have delved into elucidating its toxicological mechanisms. Studies have demonstrated that exposure of HepG2 by AA in a range of concentrations can induce the upregulation of miR-21 and miR-221. Monitoring the response of intracellular miRNAs can play an important role in unraveling the mechanisms of AA toxicity. Here, multicolor aggregation induced emission nano particle (AIENP) probes were constructed from three AIE dyes for simultaneous imaging of intracellular AA and AA-induced miR-21/miR-221 by combining the recognition function of AA aptamers and the signal amplification of a DNAzyme walker. The surface of these nanoparticles contains carboxyl groups, facilitating their linkage to a substrate chain modified with a fluorescent quencher group via an amide reaction. Optimization experiments were conducted to determine the optimal substrate-to-DNAzyme ratio, confirming its efficacy as a walker for signal amplification. Sensitive detection of AA, miR-21 and miR-221 was achieved in extracellular medium, with detection limits of 0.112 nM for AA, 0.007 pM and 0.003 pM for miR-21 and miR-221, respectively, demonstrating excellent selectivity. Intracellularly, ZIF-8 structure collapsed, releasing Zn2+, activating DNAzyme cleavage activity, and the fluorescence of multicolor AIENPs within HepG2 cells gradually recovered with increasing stimulation time (0-12 h) and concentrations of AA (0-500 µM). This dynamic response unveiled the relationship between AA exposure and miR-21/miR-221 expression, further validating the carcinogenicity of AA.

Dual-mode aptasensor based on P-CeO2NR@Mxene and exonuclease I-assisted target recycling for malachite green detection.

Malachite green (MG) has been illicitly employed in aquaculture as a parasiticide, however, its teratogenic and carcinogenic effects pose a significant human health threat. Herein, a dual-mode colorimetric and electrochemical aptasensor was fabricated for MG detection, capitalizing on the robust catalytic and peroxidase-like activity of P-CeO2NR@Mxene and good capture efficiency of a tetrahedral DNA nanostructure (TDN) designed with multiple aptamers (m-TDN). P-CeO2NR@Mxene-modified complementary DNA (cDNA) served as both colorimetric and electrochemical probe. m-TDN was attached to AuE to capture MG and P-CeO2NR@Mxene/cDNA. The superior aptamer and MG binding to cDNA regulated signals and enabled precise MG quantification. The further introduced Exo I enabled aptamer hydrolysis, releasing MG for further binding rounds, allowing target recycling amplification. Under the optimal conditions, the aptasensor reached an impressively low detection limit 95.4 pM in colorimetric mode and 83.6 fM in electrochemical mode. We believe this dual-mode approach holds promise for veterinary drug residue detection.

Rational Design of a Robust G-Quadruplex Aptamer as an Inhibitor to Alleviate Listeria monocytogenes Infection.

Listeria monocytogenes (LM) is one of the most invasive foodborne pathogens that cause listeriosis, making it imperative to explore novel inhibiting strategies for alleviating its infection. The adhesion and invasion of LM within host cells are partly orchestrated by an invasin protein internalin A (InlA), which facilitates bacterial passage by interacting with the host cell E-cadherin (E-Cad). Hence, in this work, we proposed an aptamer blocking strategy by binding to the region on InlA that directly mediated E-Cad receptor engagement, thereby alleviating LM infection. An aptamer GA8 with a robust G-quadruplex (G4) structural feature was designed through truncation and base mutation from the original aptamer A8. The molecular docking and dynamics analysis showed that the InlA/aptamer GA8 binding interface was highly overlapping with the natural InlA/E-Cad binding interface, which confirmed that GA8 can tightly and stably bind InlA and block more distinct epitopes on InlA that involved the interaction with E-Cad. On the cellular level, it was confirmed that GA8 effectively blocked LM adhesion with an inhibition rate of 78%. Overall, the robust G4 aptamer-mediated design provides a new direction for the development of inhibitors against other wide-ranging and emerging pathogens.

Fe-Co-Based Metal-Organic Frameworks as Peroxidase Mimics for Sensitive Colorimetric Detection and Efficient Degradation of Aflatoxin B1.

Building multifunctional platforms for integrating the detection and control of hazards has great significance in food safety and environment protection. Herein, bimetallic Fe-Co-based metal-organic frameworks (Fe-Co-MOFs) peroxidase mimics are prepared and applied to develop a bifunctional platform for the synergetic sensitive detection and controllable degradation of aflatoxin B1 (AFB1). On the one hand, Fe-Co-MOFs with excellent peroxidase-like activity are combined with target-induced catalyzed hairpin assembly (CHA) to construct a colorimetric aptasensor for the detection of AFB1. Specifically, the binding of aptamer with AFB1 releases the prelocked Trigger to initiate the CHA cycle between hairpin H2-modified Fe-Co-MOFs and hairpin H1-tethered magnetic nanoparticles to form complexes. After magnetic separation, the colorimetric signal of the supernatant in the presence of TMB and H2O2 is inversely proportional to the target contents. Under optimal conditions, this biosensor enables the analysis of AFB1 with a limit of detection of 6.44 pg/mL, and high selectivity and satisfactory recovery in real samples are obtained. On the other hand, Fe-Co-MOFs with remarkable Fenton-like catalytic degradation performance for organic contaminants are further used for the detoxification of AFB1 after colorimetric detection. The AFB1 is almost completely removed within 120 min. Overall, the introduction of CHA improves the sensing sensitivity; efficient postcolorimetric-detection degradation of AFB1 reduces the secondary contamination and risk to the experimental environment and operators. This strategy is expected to provide ideas for designing other multifunctional platforms to integrate the detection and degradation of various hazards.

A colorimetric and SERS dual-mode aptasensor for the detection of Shiga toxin type II based on Mn/Fe-MIL(53)@AuNSs.

Shiga toxin type II (Stx2), the major virulence component of enterohemorrhagic Escherichia coli, is strongly associated with the life-threatening hemolytic uremic syndrome thus posing a substantial risk to food safety and human health. In this work, a dual-mode aptasensor with colorimetric and surface-enhanced Raman scattering was developed for Stx2 specific detection based on noble metal nanoparticles and Raman reporter loaded metal-organic framework (Mn/Fe-MIL(53)@AuNSs-MBA). The Mn/Fe-MIL(53)@AuNSs could catalyze the H2O2-mediated oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), thereby enabling visual detection. Meanwhile, the SERS signal from MBA can be enhanced by the decorated AuNSs. Under optimal conditions, a linear range of 0.05-500 ng/mL with limit of detection (LOD) of 26 pg/mL was achieved in colorimetric mode and a linear range of 5-1000 ng/mL with LOD of 0.82 ng/mL in SERS mode, in which the dual-mode results complement each other, widening the linear range, increasing the accuracy and reliability of the detection. The method was further applied to the detection of Stx2 in milk with average recovery of 101.1 %, demonstrating its superior potential for bacterial toxin monitoring.

Generation of a specific aptamer for accurate detection of sarafloxacin based on fluorescent/colorimetric/SERS triple-readout sensor.

Sarafloxacin (SAR), one of the most widely used fluoroquinolone antibiotics, is a serious threat to aquatic environments and human health due to its illegal abuse. Herein, we first screened an aptamer (SAR-1) that specifically binds to SAR using capture-SELEX technology. Based on molecular docking technology, SAR-1 was gradually truncated, and a short SAR-1a with better affinity and specificity was obtained. The optimal SAR-1a was further combined with a Pt nanoparticle (Pt NP)- decorated bimetallic Fe/Co-MOF to fabricate a multimode sensing platform for SAR determination. The Fe/Co-MOF@Pt NPs exhibited excellent peroxidase-like activity, which catalyzed the H2O2-mediated oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), thereby enabling visual detection of SAR. Meanwhile, the generated oxTMB can also produce SERS responses and be used for the SERS detection of SAR. Moreover, the inherent fluorescence property of Fe/Co-MOF@Pt NPs enabled fluorescence detection of SAR. The designed triple-readout aptasensor showed good sensitivity for SAR detection with limits of detection of 0.125 ng/mL (fluorescent mode) and 0.05 ng/mL (colorimetric and SERS mode). The aptamer-based triple-mode sensing platform provided mutual verification of detection results in different output modes, effectively improving the assay accuracy and providing a promising tool for highly sensitive, selective, and accurate determination of SAR in daily life.

Detection of acrylamide in food based on MIL-glucose oxidase cascade colorimetric aptasensor.

BACKGROUND: Maillard reaction involves the polymerization, condensation, and other reactions between compounds containing free amino groups and reducing sugars or carbonyl compounds during heat processing. This process endows unique flavors and colors to food, while it can also produce numerous hazards. Acrylamide (AAm) is one of Maillard's hazards with neurotoxicity and carcinogenicity, these effects can trigger mutations and alternations in gene expression in human cells and accelerate organ aging. An accurate and reliable acrylamide detection method with high sensitivity and specificity for future regulatory activities is urgently needed. RESULTS: Herein, we constructed a colorimetric aptasensor with the hybridization of MIL-glucose oxidase (MGzyme)-cDNA and magnetic nanoparticle-aptamer (MNP-Apt) to specifically detect AAm. The incorporation of MB-Apt and AAm released MGzyme-cDNA in the supernatant, took the supernatant out, with the addition of glucose and TMB, MGzyme would oxidize glucose, the resulting •OH facilitated the oxidation of colorless TMB to blue ox-TMB. The absorbance value at 652 nm, which indicates the characteristic absorption peak of ox-TMB, exhibited a proportion to the concentration of AAm. MGzyme avoided the addition of harmful intermediate H2O2 and created an acid microenvironment for the catalytic reaction. MNP-Apt possessed the advantages of high specificity and simplified separation. Under optimal conditions, this method displayed a linear range of 0.01-100 µM with the limit of detection of 1.53 nM. With the spiked analysis data cross-verified by ELISA kit, this aptasensor was proven to specifically detect AAm at low concentrations. SIGNIFICANCE: This colorimetric aptasensor was the integration of aptamer and the enzyme-cascade system, which could broaden the applicable range of enzyme-cascade system, break the limits of specific detection of substrates, eliminate the need for harmful intermediates, improve the reaction efficiency, implement the specific detection, whilst enabling the accurate detection of AAm. Given these remarkable performances, this method has shown significant potential in the field of food safety inspection.

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.

Ratiometric SERS aptasensing for simultaneous quantitative detection of histamine and tyramine in fishes.

Herein, a SiO2@Ag NPs core/shell nanoparticles were synthesized to fabricate a surface-enhanced Raman spectroscopy (SERS) sensor for the simultaneous determination of histamine (HIS) and tyramine (TYR) based on specific aptamer recognition and ratiometric strategy. SiO2@Ag NPs with 4-thiosaminophenol (4-ATP) and Nile blue A (NBA) molecules were used as an internal standard (IS) and labeled with aptamers corresponding to HIS and TYR, respectively. Raman reporter molecules ROX and Cy5 labeled complementary DNA (cDNA) were then hybridized with aptamers to form rigid double-stranded DNA. After the HIS and TYR were captured by their aptamers, resulting in the dissociation of cDNA and separated from the SERS substrate. Therefore, the SERS signal intensity at 1503 cm-1 of ROX and 1358 cm-1 of Cy5 tagged on the terminal of cDNA decreased with the concentration of HIS and TYR increasing, while the SERS signal intensity at 1079 cm-1 of 4-APT and 592 cm-1 of NBA on the substrate remain stable. Thus, the concentrations of HIS and TYR can be determined by the I1503/I1079 and I1358/I592 values, respectively. This sensing strategy achieves a lower detection limit of 0.2 ng/mL for HIS and 0.05 ng/mL for TYR, respectively, demonstrating promising applications in sensitive detection of BAs in animal-derived foodstuff.

A fluorescence and surface-enhanced Raman scattering dual-mode aptasensor for sensitive detection of deoxynivalenol based on gold nanoclusters and silver nanoparticles modified metal-polydopamine framework.

Deoxynivalenol (DON), a common mycotoxin produced by Fusarium species, poses a great threat to human and animal body. Hence, it is of significance to develop an ultrasensitive and reliable method for DON detection. Herein, a fluorescence and surface-enhanced Raman scattering (FL-SERS) dual-mode aptasensor was designed for the detection of DON based on gold nanoclusters (Au NCs) and silver nanoparticles modified metal-polydopamine framework (Ag NPs/MPDA). In this aptasensor, complementary DNA modified Au NCs (cDNA-Au NCs) was selected as fluorescence probe, and 6-carboxytetramethylrhodamine (TAMRA)-labeled aptamer modified Ag NPs/MPDA (Ag NPs/MPDA-Apt-TAMRA) was employed as SERS probe, in which Ag NPs/MPDA acted as SERS substance and fluorescence quencher, and TAMRA acted as Raman label. The superior binding affinity of the aptamer with DON to cDNA can regulate the fluorescence and Raman signal intensities and realize the quantitative determination of DON. Under the optimal conditions, the aptasensor exhibited a low detection limit of 0.08 ng mL-1 (0.1-100 ng mL-1) in FL mode and 0.06 ng mL-1 (0.1-100 ng mL-1) in SERS mode. In addition, it was successfully applied for DON detection in wheat flour. We believe that the proposed FL-SERS strategy has a promising application in the detection of mycotoxins.

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.

A sensitive fluorescent assay based on gold-nanoclusters coated on molecularly imprinted covalent organic frameworks and its application in malachite green detection.

Malachite green (MG), as a parasiticide, is widely used in aquaculture to increase the production of the fishery industry. It poses a great danger to both the food system and the human body. In this study, a one-pot reverse microemulsion polymerization was employed to combine the gold nanoclusters (AuNCs) with molecularly imprinted polymers (MIPs) and covalent organic frameworks (COFs) to synthesize an efficient fluorescent hybrid probe (AuNCs@COFs@MIPs) for selective detection of MG. The specific recognition of AuNCs@COFs@MIPs towards MG triggers the fluorescence quenching of AuNCs. The fluorescent response was linearly related to the concentration over the range of 10-150 nmol/L with a limit of detection of 2.78 nmol/L. In addition, the proposed probe was further applied to fish and water samples. A favorable recovery ranged from 97.34 to 101.51 % toward trace amounts of MG indicating its promising application for detecting residue of veterinary drugs.

Screening of specific aptamers against chlorpromazine and construction of novel ratiometric fluorescent aptasensor based on metal-organic framework.

Chlorpromazine is a phenothiazine representative drug that can be used to anesthetize and calm animals. However, chlorpromazine excess may lead to residual persistence in edible tissues, which is potentially harmful to human health and animal production. In this work, high affinity and specificity aptamers against chlorpromazine were screened out based on Capture-SELEX. After ten rounds of screening, the candidate aptamers were obtained. The optimal aptamer of CHL-3 was obtained by isothermal titration calorimetry (ITC) and SYBR Green I (SGI) fluorescence-competition methods. The Kd value of CHL-3 was 69.8 ± 9.81 nM. Subsequently, the Uio-66-NH2 material was prepared, filled with rhodamine 6G (Rho 6G) dye into the pore, and sealed with CHL-3, and fluorescence probes were obtained. The ratiometric fluorescence detection method was established to detect the concentration of chlorpromazine. A linear relationship was obtained in a range of 1-100 nM, with a lower detection limit of 0.67 nM. Meanwhile, a good recovery was shown in spiked food samples, such as eggs and milk. These results indicate that the constructed ratiometric fluorescence strategy can be successfully applied in food detection.

A portable paper-based aptasensor for simultaneous visual detection of two mycotoxins in corn flour using dual-color upconversion nanoparticles and Cu-TCPP nanosheets.

A portable paper-based microfluidic aptasensor is established to simultaneously and visually detect zearalenone (ZEN) and ochratoxin A (OTA). The targets at the sample zone can migrate to two detection zones through dual-channels and result in green and blue fluorescence recovery. This is due to the specific recognition by a respective aptamer that destroys fluorescence resonance energy transfer (FRET) from dual-color upconversion nanoparticles (UCNPs) to Cu-TCPP nanosheets. By capturing fluorescent images and analyzing the corresponding RGB value via a smartphone, ZEN and OTA can be analyzed with limits of detection down to 0.44 ng/mL and 0.098 ng/mL in the linear ranges of 0.5-100 ng/mL and 0.1-50 ng/mL, respectively. Satisfactory recoveries are also obtained for ZEN (94.5-103.7 %) and OTA (92.2-106.8 %) in corn flour. With the advantages of simple operation, low sample consumption, and broad adaptability, this promising platform allows for the on-site detection of multiple hazards in food.

Selection of aptamer targeting levamisole and development of a colorimetric and SERS dual-mode aptasensor based on AuNPs/Cu-TCPP(Fe) nanosheets.

Levamisole (LEV) is a veterinary drug that often remains in animal food. Consuming products containing high levels of LEV will cause a series of harmful reactions to human health. This work describes the Capture-SELEX (Capture-systematic evolution of ligands by exponential enrichment) screening strategy of LEV aptamers, using streptavidin modified agarose beads as a solid phase medium to separate target-bound and unbound ssDNA. The affinity and specificity of candidate aptamers were determined by SYBR Green I (SGI) dye and isothermal titration calorimetry (ITC), in which LEV-5 showed good binding affinity and specificity, and the dissociation constant was 66.15 ± 11.86 nM. Circular dichroism (CD) was used to characterize aptamer conformational changes before and after target binding, including increased helicity and enhanced base stacking. To evaluate whether this aptamer can be used for LEV detection, a colorimetric-surface-enhanced Raman spectroscopy (colorimetric-SERS) dual-mode aptasensor was constructed based on the peroxidase-like activity and SERS effect of AuNPs/Cu-TCPP(Fe) nanosheets. The detection limits of this dual-mode aptasensor for LEV were 5 nM and 1.12 nM, respectively. This aptamer-based method was further successfully used to detect LEV in milk, with recoveries ranging from 94.95% to 111.2%, providing a potential application for the detection of harmful substances in food.

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.

Laser-Printed Paper-Based Microfluidic Chip Based on a Multicolor Fluorescence Carbon Dot Biosensor for Visual Determination of Multiantibiotics in Aquatic Products.

Excessive use of antibiotics in aquaculture severely endangers human health and ecosystems, which has raised significant concerns in recent years. However, conventional laboratory-based approaches regularly required time or skilled manpower. Herein, we propose a point-of-care-testing (POCT) biosensor detection device for the simultaneous determination of multiantibiotics without complex equipment or professional operators. A laser-printed paper-based microfluidic chip loaded with multicolor fluorescence nanoprobes (mCD-µPAD) was developed to rapidly detect sulfamethazine (SMZ), oxytetracycline (OTC), and chloramphenicol (CAP) on-site. These "fluorescence off" detection probes composed of carbon dots (CDs) conjugated with aptamers (donor) and MoS2 nanosheets (acceptor) (CD-apt-MoS2) were based on Förster resonance energy transfer. Upon the addition of target antibiotics, the significantly recovered fluorescence signal on the µPAD can be sensitively perceived by employing a 3D-printed portable detection box through a smartphone. Under optimal conditions, this µPAD allowed for a rapid response of 15 min toward SMZ, OTC, and CAP with considerable sensitivities of 0.47, 0.48, and 0.34 ng/mL, respectively. In shrimp samples, the recoveries were 95.2-101.2, 96.4-105, and 96.7-106.1% with RSD below 6%. This paper-based sensor opens an avenue for on-site, high-throughput, and rapid detection methods and can be widely used in POCT in food safety.