Search

Tylosin (TYL) is a macrolide antibiotic widely used in animal husbandry. Due to associated health risks, there is a demand for sensitive methods for mass screening of TYL in products of animal origin. This article describes the development of lateral flow immunoassays (LFIAs) for TYL detection using direct (anti-TYL antibodies conjugated with nanoparticles) and indirect antibody labeling (anti-species antibodies conjugated with nanoparticles and combined with native anti-TYL antibodies). The choice of LFIA conditions, such as concentrations of hapten–protein conjugates, specific antibodies, and gold nanoparticle (GNP) conjugates with antibodies, as well as incubation time of reagents and the concentration of detergent in the sample buffer, is presented. The achieved limits of TYL detection using LFIAs with indirect labeling were 0.8 ng/mL (visual) and 0.07 ng/mL (instrumental), compared to 4 ng/mL (visual) and 0.4 ng/mL (instrumental) for the case of direct labeling. The sensitivity of the LFIA using the indirect format was up to seven times higher, allowing the determination of the target analyte at low concentrations. TYL detection in ground meat using LFIA with indirect antibody labeling ranged from 76–119%. [ABSTRACT FROM AUTHOR]

Salmonella typhimurium and Listeria monocytogenes are relevant foodborne bacterial pathogens which may cause serious intoxications and infectious diseases in humans. In this study, a sensitive immunochromatographic analysis (ICA) for the simultaneous detection of these two pathogens was developed. For this, test strips containing two test zones with specific monoclonal antibodies (MAb) against lipopolysaccharides of S. typhimurium and L. monocytogenes and one control zone with secondary antibodies were designed, and the double-assay conditions were optimized to ensure high analytical parameters. Prussian blue nanoparticles (PBNPs) were used as nanozyme labels and were conjugated with specific MAbs to perform a sandwich format of the ICA. Peroxidase-mimic properties of PBNPs allowed for the catalytic amplification of the colorimetric signal on test strips, enhancing the assay sensitivity. The limits of detection (LODs) of Salmonella and Listeria cells were 2 × 102 and 7 × 103 cells/mL, respectively. LODs were 100-fold less than those achieved due to the ICA based on the traditional gold label. The developed double ICA was approbated for the detection of bacteria in cow milk samples, which were processed by simple dilution by buffer before the assay. For S. typhimurium and L. monocytogenes, the recoveries from milk were 86.3 ± 9.8 and 118.2 ± 10.5% and correlated well with those estimated by the enzyme-linked immunosorbent assay as a reference method. The proposed approach was characterized by high specificity: no cross-reactivity with other bacteria strains was observed. The assay satisfies the requirements for rapid tests: a full cycle from sample acquisition to result assessment in less than half an hour. The developed ICA has a high application potential for the multiplex detection of other foodborne pathogens. [ABSTRACT FROM AUTHOR]

A promising and sought-after class of nanozymes for various applications is Pt-containing nanozymes, primarily Au@Pt, due to their easy preparation and remarkable catalytic properties. This study aimed to explore the freeze–thaw method for functionalizing Pt-containing nanozymes with oligonucleotides featuring a polyadenine anchor. Spherical gold nanoparticles ([Au]NPs) were synthesized and subsequently used as seeds to produce urchin-like Au@Pt nanoparticles ([Au@Pt]NPs) with an average diameter of 29.8 nm. The nanoparticles were conjugated with a series of non-thiolated DNA oligonucleotides, each consisting of three parts: a 5′-polyadenine anchor (An, with n = 3, 5, 7, 10; triple-branched A3, or triple-branched A5), a random sequence of 23 nucleotides, and a linear polyT block consisting of seven deoxythymine residues. The resulting conjugates were characterized using transmission electron microscopy, spectroscopy, dynamic light scattering, and emission detection of the fluorescent label at the 3′-end of each oligonucleotide. The stability of the conjugates was found to depend on the type of oligonucleotide, with decreased stability in the row of [Au@Pt]NP conjugates with A7 > A5 > 3A3 > 3A5 > A10 > A3 anchors. These [Au@Pt]NP–oligonucleotide conjugates were further evaluated using lateral flow test strips to assess fluorescein-specific binding and peroxidase-like catalytic activity. Conjugates with A3, A5, A7, and 3A3 anchors showed the highest levels of signals of bound labels on test strips, exceeding conjugates in sensitivity by up to nine times. These findings hold significant potential for broad application in bioanalytical systems. [ABSTRACT FROM AUTHOR]

An immunochromatographic test system based on the indirect competitive format has been developed for the rapid control of cyanotoxin microcystin-LR (MC-LR) in drinking water. To increase the assay sensitivity, a hydroquinone-based reduction of silver ions on the surface of gold nanoparticles as part of the immune complexes formed on the immunochromatographic membrane has been carried out. This stage allows for the enhancement of the colouration in test strip zones. The developed test system is characterised by an instrumental limit of detection of 0.05 ng/mL, a cut-off of 0.5 ng/mL, and a working range of the detectable concentrations of 0.08–0.42 ng/mL. The assay duration was 17 min. The applicability of the test system for MC-LR detection in drinking water is demonstrated. The assay recoveries vary from 90% to 120%. [ABSTRACT FROM AUTHOR]

Fluorescence induced by the excitation of a fluorophore with plane-polarized light has a different polarization depending on the size of the fluorophore-containing reagent and the rate of its rotation. Based on this effect, many analytical systems have been implemented in which an analyte contained in a sample and labeled with a fluorophore (usually fluorescein) competes to bind to antibodies. Replacing antibodies in such assays with aptamers, low-cost and stable oligonucleotide receptors, is complicated because binding a fluorophore to them causes a less significant change in the polarization of emissions. This work proposes and characterizes the compounds of the reaction medium that improve analyte binding and reduce the mobility of the aptamer–fluorophore complex, providing a higher analytical signal and a lower detection limit. This study was conducted on aflatoxin B1 (AFB1), a ubiquitous toxicant contaminating foods of plant origins. Eight aptamers specific to AFB1 with the same binding site and different regions stabilizing their structures were compared for affinity, based on which the aptamer with 38 nucleotides in length was selected. The polymers that interact reversibly with oligonucleotides, such as poly-L-lysine and polyethylene glycol, were tested. It was found that they provide the desired reduction in the depolarization of emitted light as well as high concentrations of magnesium cations. In the selected optimal medium, AFB1 detection reached a limit of 1 ng/mL, which was 12 times lower than in the tris buffer commonly used for anti-AFB1 aptamers. The assay time was 30 min. This method is suitable for controlling almond samples according to the maximum permissible levels of their contamination by AFB1. The proposed approach could be applied to improve other aptamer-based analytical systems. [ABSTRACT FROM AUTHOR]

In this study, a fluorescence resonance energy transfer (FRET)-based aptasensor for the detection of aflatoxin B1 (AFB1) was designed using a carboxyfluorescein (FAM)-labeled aptamer and short complementary DNA (cDNA) labeled with low molecular quencher RTQ1. The sensing principle was based on the detection of restored FAM-aptamer fluorescence due to the ligand-induced displacement of cDNA in the presence of AFB1, leading to the destruction of the aptamer/cDNA duplex and preventing the convergence of FAM and RTQ1 at the effective FRET distance. Under optimal sensing conditions, a linear correlation was obtained between the fluorescence intensity of the FAM-aptamer and the AFB1 concentration in the range of 2.5–208.3 ng/mL with the detection limit of the assay equal to 0.2 ng/mL. The assay time was 30 min. The proposed FRET aptasensor has been successfully validated by analyzing white wine and corn flour samples, with recovery ranging from 76.7% to 91.9% and 84.0% to 86.5%, respectively. This work demonstrates the possibilities of labeled cDNA as an effective and easily accessible tool for sensitive AFB1 detection. The homogeneous FRET aptasensor is an appropriate choice for contaminant screening in complex matrices. [ABSTRACT FROM AUTHOR]