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When developing immunochemical test systems, it is necessary to obtain specific antibodies. Their quality depends, among other things, on the immunogen used. When preparing hapten–protein conjugates to obtain antibodies for low-molecular-weight compounds, the key factors are the structure of the hapten itself, the presence of a spacer, the size of the carrier protein and the degree of its modification by hapten molecules. This work shows that one additional factor—the conditions for obtaining the hapten–protein conjugate—is overlooked. In this work, we have synthesized conjugates of bisphenol A derivative 4,4-bis(hydroxyphenyl)valeric acid (BVA), the protein carrier soybean trypsin inhibitor (STI), and bovine serum albumin (BSA) in reaction media combining water with two organic solvents: dimethylformamide (DMF) or dimethyl sulfoxide (DMSO). Namely, BSADMF–BVA, STIDMF–BVA, BSADMSO–BVA and STIDMSO–BVA conjugates were obtained. Rabbit polyclonal antibodies against the BSADMF–BVA conjugate demonstrated basically different interactions in the developed ELISA systems using either STIDMF–BVA or STIDMSO–BVA conjugates. The use of the STIDMF–BVA conjugate demonstrated the absence of competition in combination with antisera obtained from BSADMF–BVA in an ELISA. A competitive interaction was observed only with the use of the STIDMSO–BVA conjugate. Under the selected conditions, the detection limit of bisphenol A was 8.3 ng/mL, and the working range of determined concentrations was 18.5–290.3 ng/mL. The obtained data demonstrate the possibility of achieving sensitive immunoassays by simply varying the reaction media for the hapten–protein conjugation, which could provide an additional tool in the development of immunoassays for other low-molecular-weight compounds. [ABSTRACT FROM AUTHOR]

Uncontrolled use of pesticides in agriculture leads to negative consequences for the environment, as well as for human and animal health. Therefore, timely detection of pesticides will allow application of measures to eliminate the excess of maximum residue limits and reduce possible negative consequences in advance. Common methods of pesticide analysis suffer from high costs, and are time consuming, and labor intensive. Currently, more attention is being paid to the development of surface-enhanced Raman scattering (SERS) sensors as a non-destructive and highly sensitive tool for detecting various chemicals in agricultural applications. This review focuses on the current developments of biocompatible SERS substrates based on natural materials with unique micro/nanostructures, flexible SERS substrates based on biopolymers, as well as functionalized SERS substrates, which are close to the current needs and requirements of agricultural product quality control and environmental safety assessment. The impact of herbicides on the process of photosynthesis is considered and the prospects for the application of Raman spectroscopy and SERS for the detection of herbicides are discussed. [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]

CRISPR/Cas12a is a potent biosensing tool known for its high specificity in DNA analysis. Cas12a recognizes the target DNA and acquires nuclease activity toward single-stranded DNA (ssDNA) probes. We present a straightforward and versatile approach to transforming common Cas12a-cleavable DNA probes into enhancing tools for fluorescence anisotropy (FA) measurements. Our study involved investigating 13 ssDNA probes with linear and hairpin structures, each featuring fluorescein at one end and a rotation-slowing tool (anchor) at the other. All anchors induced FA changes compared to fluorescein, ranging from 24 to 110 mr. Significant FA increases (up to 180 mr) were obtained by adding divalent metal salts (Mg2+, Ca2+, Ba2+), which influenced the rigidity and compactness of the DNA probes. The specific Cas12a-based recognition of double-stranded DNA (dsDNA) fragments of the bacterial phytopathogen Erwinia amylovora allowed us to determine the optimal set (probe structure, anchor, concentration of divalent ion) for FA-based detection. The best sensitivity was obtained using a hairpin structure with dC10 in the loop and streptavidin located near the fluorescein at the stem in the presence of 100 mM Mg2+. The detection limit of the dsDNA target was equal to 0.8 pM, which was eight times more sensitive compared to the common fluorescence-based method. The enhancing set ensured detection of single cells of E. amylovora per reaction in an analysis based on CRISPR/Cas12a with recombinase polymerase amplification. Our approach is universal and easy to implement. Combining FA with Cas12a offers enhanced sensitivity and signal reliability and could be applied to different DNA and RNA analytes. [ABSTRACT FROM AUTHOR]

The toxic effects of antimony pose risks to human health. Therefore, simple analytical techniques for its widescale monitoring in water sources are in demand. In this study, a sensitive microplate apta-enzyme assay for Sb3+ detection was developed. The biotinylated aptamer A10 was hybridized with its complementary biotinylated oligonucleotide T10 and then immobilized on the surface of polysterene microplate wells. Streptavidin labeled with horseradish peroxidase (HRP) bound to the biotin of a complementary complex and transformed the 3,3′,5,5′-tetramethylbenzidine substrate, generating an optical signal. Sb3+ presenting in the sample bounded to an A10 aptamer, thus releasing T10, preventing streptavidin-HRP binding and, as a result, reducing the optical signal. This effect allowed for the detection of Sb3+ with a working range from 0.09 to 2.3 µg/mL and detection limit of 42 ng/mL. It was established that the presence of Ag+ at the stage of A10/T10 complex formation promoted dehybridization of the aptamer A10 and the formation of the A10/Sb3+ complex. The working range of the Ag+-enhanced microplate apta-enzyme assay for Sb3+ was determined to be 8–135 ng/mL, with a detection limit of 1.9 ng/mL. The proposed enhanced approach demonstrated excellent selectivity against other cations/anions, and its practical applicability was confirmed through an analysis of drinking and spring water samples with recoveries of Sb3+ in the range of 109.0–126.2% and 99.6–106.1%, respectively. [ABSTRACT FROM AUTHOR]

In this study, tannic acid-modified gold nanoparticles were found to have superior nanozyme activity and catalyze the oxidation reaction of 3,3′,5,5′-tetramethylbenzidine in the presence of hydrogen peroxide. Enhancing the catalytic activity of the nanozyme by Pb2+ ions caused by selectively binding metal ions by the tannic acid-capped surface of gold nanoparticles makes them an ideal colorimetric probe for Pb2+. The parameters of the reaction, including pH, incubation time, and concentration of components, were optimized to reach maximal sensitivity of Pb2+ detection. The absorption change is directly proportional to the Pb2+ concentration and allows the determination of Pb2+ ions within 10 min. The colorimetric sensor is characterized by a wide linear range from 25 to 500 ng×mL−1 with a low limit of detection of 11.3 ng×mL−1. The highly sensitive and selective Pb2+ detection in tap, drinking, and spring water revealed the feasibility and applicability of the developed colorimetric sensor. [ABSTRACT FROM AUTHOR]