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Abscisic acid (ABA) is a phytohormone that occurs in plants at very low concentration (pmol/g fresh weight) and regulates multiple biological processes, including stomatal closure, seed germination, and responses to environmental stresses. In the present study, isolation of ABA, ABA glucosyl ester, and 11 ABA amino acid conjugates from minute quantities of plant tissue (less than 20 mg fresh weight) was achieved using a purification method based on the combination of an Oasis HLB column and an immunoaffinity sorbent. New monoclonal antibodies raised against (+)-cis,trans-ABA conjugated to BSA through its carboxyl group (C1) were characterised by enzyme-linked immunosorbent assay (ELISA) and used for immunoaffinity chromatography (IAC) gel preparation. The use of immunoaffinity purification significantly reduced matrix effects and increased the selectivity and sensitivity of subsequent UHPLC-MS/MS analysis. In addition to (+)-cis,trans-ABA and its glucosyl ester, a new abscisic acid conjugate, ABA-L-glutamate, was isolated by IAC and identified by tandem mass spectrometry in pea (Pisum sativum L.), Lepidium sativum L. and wheat (Triticum aestivum L.) seedlings. However, it was not found in 10-day-old seedlings of Arabidopsis thaliana or water-stressed tobacco (Nicotiana tabacum L.) leaves. Here, the identification of an ABA conjugate with glutamic acid in plants is described for the first time.

Liquid chromatography (LC) coupled with mass spectrometry (MS) is widely used for the determination of mycotoxins in cereals and cereal-based products. In addition to the regulated mycotoxins, for which official control is required, LC–MS is often used for the screening of a large range of mycotoxins and/or for the identification and characterization of novel metabolites. This review provides insight into the LC–MS methods used for the determination of co-occurring mycotoxins with special emphasis on multiple-analyte applications. The first part of the review is focused on targeted LC–MS approaches using cleanup methods such as solid-phase extraction and immunoaffinity chromatography, as well as on methods based on minimum cleanup (quick, easy, cheap, effective, rugged, and safe; QuEChERS) and dilute and shoot. The second part of the review deals with the untargeted determination of mycotoxins by LC coupled with high-resolution MS, which includes also metabolomics techniques to study the fate of mycotoxins in plants.

Deoxynivalenol (DON) and zearalenone (ZEN) are mycotoxins that contaminate a wide range of grains and crops. In this study, a one-step time-resolved single-channel immunochromatographic test strip based on europium ion polystyrene fluorescence microspheres was first developed for sensitive and quantitative detection of DON and ZEN. The concentration of the artificial antigen and the mass ratio of the monoclonal antibody to fluorescent microspheres for conjugation were optimized to simplify the sample addition process during immunochromatographic assay and improve the on-site detection efficiency. The limits of detection (LOD) of the single-channel immunochromatographic test strip for DON and ZEN detection were 0.17 and 0.54 μg/L, respectively. Meanwhile, the dual-channel immunochromatographic test strip was designed to simultaneously detect DON and ZEN, with LODs of 0.24 and 0.69 μg/L achieved for DON and ZEN, respectively. The developed test strips also yielded recovery results consistent with that obtained by LC-MS/MS for DON and ZEN detection in real samples of wheat and corn flour, confirming the practicability and reliability of the test strip. The developed immunochromatographic test strips realize quick and sensitive detection of DON and ZEN, exhibiting potential for broad applications in the point-of-care testing platform of multiple mycotoxins in agricultural products.

Extracellular vesicles (EVs) are nanosized structures involved in intercellular communication that have high potential as disease biomarkers and for the delivery of therapeutic cargos. However, translation to the clinic is hampered by time-consuming, low-yield, and poorly reproducible EV isolation methods. We describe a cryogel-based immunoaffinity chromatography system that exploits single-domain VHH antibodies as capture elements for the selective isolation of EVs from human plasma. Supermacroporous cryogels functionalized with five unique anti-EV VHHs (total immobilization capacity ~500 µg/g) were prepared, yielding a highly permeable and hydrophilic support. They were captured and eluted under mild conditions, and their morphology and identity were confirmed by SEM, AFM, NTA, and flow cytometry. Proteomic profiling of the isolated samples identified 234 proteins, of which 63% were ExoCarta-listed exosomal proteins; contaminants such as albumin and apolipoproteins were also identified. The purification method provided samples with ~2 × 109 EVs/mL, with EV median size of 135 nm and consistent protein-to-lipid ratio across three independent isolations (CV < 10%). This study demonstrates that VHH-functionalized cryogels (VHH-SMC) are a rapid and reproducible EV purification method that represents a promising alternative to conventional ultracentrifugation- or precipitation-based protocols. While optimization of nanobody density and reduction in plasma protein carryover are still necessary, the platform holds potential for scalable EV enrichment, a condition that can significantly speed up biomarker research and clinical diagnostics.

Paper has been present in the world of analytical chemistry for centuries, but it seems that just a few years back it was rediscovered as a valuable substrate for sensors. We can easily list some of the countless advantages of this simple cellulosic substrate, including mechanical properties, three-dimensional fibrous structure, biocompatibility and biodegradability, easiness of production and modification, reasonable price, and availability all over the world. Those characteristics make paper a first-choice substrate for disposable sensors and integrated sensing platforms. Nowadays, numerous examples of paper-based sensors are being presented in the literature. This review describes some of the most prominent examples classifying them by type of detection: optical (colorimetric, fluorescence, surface-enhanced Raman spectroscopy, and transmittance methods) and electrochemical (voltammetric, potentiometric, and conductivity-based methods). We take a closer look at recent advances in immunoassays fabricated on paper, excluding simple lateral flow tests assembled on nitrocellulose. This review also summarizes the main advantages and disadvantages of the use of paper as a substrate for sensors, as well as its impact on their performance and application, presents a short history of paper in analytical chemistry, and discusses fabrication methods and available sources of paper.

Histamine, a biogenic amine, is abundant in fermented foods and beverages, notably wine. A high intake of this monoamine may produce adverse reactions in humans, which may be severe in individuals with a reduced capacity to catabolise extrinsic histamine. Thus, control of histamine concentration during wine production and before distribution is advisable. Simple, rapid, point-of-use bioanalytical platforms are needed because traditional methods for the detection and quantification of histamine are expensive and time-consuming. This work applies the lateral flow immunoassay technique to histamine detection. Superparamagnetic particle labels, and an inductive sensor designed to read the test line in the immunoassay, enable magnetic quantification of the molecule. The system is calibrated with histamine standards in the interval of interest for wine production. A commercial optical strip reader is used for comparison measurements. The lateral flow system has a limit of detection of 1.2 and 1.5 mg/L for the inductive and optical readers, respectively. The capability of the inductive system for histamine quantification is demonstrated for wine samples at different processing points (at the end of alcoholic fermentation, at the end of malolactic fermentation, in freshly bottled wine, and in reserve wine). The results are validated by ultra-high-performance liquid chromatography.

Due to universal contamination and synergistic toxicity of multiple mycotoxins in foodstuff, reliable and high-throughput detection methods for multiple mycotoxins are urgently needed in corn products. In this study, a novel dual-channel immunochromatographic assay (ICA) based on improved up-conversion nanoparticles (IUCNPs) was developed for rapidly detecting aflatoxin B1 (AFB1) and zearalenone (ZEN). The synthesized IUCNPs doped by 30% Lu3+ showed a larger size, more regular structure, and brighter fluorescence intensity than conventional UCNPs. The limits of detection (LODs) of single-channel ICA test strips for AFB1 and ZEN detection were 0.01 and 0.1 ng/mL, respectively. After the optimization, the dual-channel ICA of AFB1 and ZEN in 10 min was conducted, resulting in low detection limits of 0.025 and 0.1 ng/mL, respectively. Moreover, the built assay was revealed to be highly specific for six other food-contaminated mycotoxins, and exhibited excellent accuracy, with corresponding R2 of 0.9931 and 0.9982 in calibration curves, respectively. Long-term storage experiments indicated that the dual-channel test strips had superior stability and precision. The LODs of AFB1 and ZEN in spiked maize were 0.025 and 0.25 μg/kg, demonstrating great sensitivity and matrix tolerance. Furthermore, the IUNCP-ICA was validated by high-performance liquid chromatography (HPLC) analyses, and a satisfactory consistency was obtained in 15 natural maize samples. Thus, the IUCNPs-ICA proposed in this work realized rapid and sensitive detection of AFB1 and ZEN, providing broad application potential in on-site screening for multiple mycotoxins in agricultural products.

African swine fever virus (ASFV) causes a highly contagious and often lethal swine viral disease, and leads to tremendous economic losses to the swine industry. Unfortunately, there are no vaccines and effective antiviral agents available to prevent and control ASFV outbreaks. Therefore, it is necessary to develop simple and rapid strategies to monitor ASFV-infected pigs to restrain its spread. In the current study, ASFV capsid protein p72 was expressed along with its chaperone pB602L to form trimers in human embryonic kidney 293 (HEK293) cells. The p72 trimers were subsequently labeled with colloidal gold to develop a immunochromatographic strip. The strip showed high specificity to ASFV-positive serum and no cross-reactivity to other swine virus positive sera. Importantly, the strip showed a higher sensitivity of detecting ASFV antibodies in both positive standard serum and clinical serum samples than a commercial enzyme-linked immunosorbent assay (ELISA) kit. Taken together, these results demonstrate the strip as a reliable diagnostic tool against ASFV infection, which will be appropriate for application in prevention and control of ASFV. Key points • ASFV p72 trimers were successfully generated . • A colloidal gold strip was developed based on ASFV p72 trimers . • The strip is appropriate for detecting ASFV antibodies in the field .

Affinity monolith chromatography (AMC) is a type of liquid chromatography that uses a monolithic support and a biologically related binding agent as a stationary phase. AMC is a powerful method for the selective separation, analysis, or study of specific target compounds in a sample. This review discusses the basic principles of AMC and recent developments and applications of this method, with particular emphasis being given to work that has appeared in the last 5 years. Various materials that have been used to prepare columns for AMC are examined, including organic monoliths, silica monoliths, agarose monoliths, and cryogels. These supports have been used in AMC for formats that have ranged from traditional columns to disks, microcolumns, and capillaries. Many binding agents have also been employed in AMC, such as antibodies, enzymes, proteins, lectins, immobilized metal ions, and dyes. Some applications that have been reported with these binding agents in AMC are bioaffinity chromatography, immunoaffinity chromatography or immunoextraction, immobilized-metal-ion affinity chromatography, dye–ligand affinity chromatography, chiral separations, and biointeraction studies. Examples are presented from fields that include analytical chemistry, pharmaceutical analysis, clinical testing, and biotechnology. Current trends and possible directions in AMC are also discussed.

Aflatoxins have posed serious threat to food safety and human health. Therefore, it is important to detect aflatoxins in samples rapidly and accurately. In this review, various technologies to detect aflatoxins in food are discussed, including conventional ones such as thin-layer chromatography (TLC), high performance liquid chromatography (HPLC), enzyme linked immunosorbent assay (ELISA), colloidal gold immunochromatographic assay (GICA), radioimmunoassay (RIA), fluorescence spectroscopy (FS), as well as emerging ones (e.g., biosensors, molecular imprinting technology, surface plasmon resonance). Critical challenges of these technologies include high cost, complex processing procedures and long processing time, low stability, low repeatability, low accuracy, poor portability, and so on. Critical discussion is provided on the trade-off relationship between detection speed and detection accuracy, as well as the application scenario and sustainability of different technologies. Especially, the prospect of combining different technologies is discussed. Future research is necessary to develop more convenient, more accurate, faster, and cost-effective technologies to detect aflatoxins. Graphical abstract