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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 (Mg 2+ , Ca 2+ , Ba 2+ ), 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 Mg 2+ . 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.

The use of a large amount of toxic synthetic materials leads to an increase in the pollution of environmental objects. Phthalates are compounds structurally related to esters of phthalic acid that are widely used in the manufacturing of synthetic packaging materials as plasticizers. Their danger is conditioned by leaching into the environment and penetrating into living organisms with negative consequences and effects on various organs and tissues. This work presents the first development of lateral flow immunoassay to detect dibutyl phthalate, one of the most common representatives of the phthalates group. To form a test zone, a hapten-protein conjugate was synthesized, and gold nanoparticles conjugated with antibodies to dibutyl phthalate were used as a detecting conjugate. The work includes the preparation of immunoreagents, selectivity investigation, and the study of the characteristics of the medium providing a reliable optical signal. Under the selected conditions for the analysis, the detection limit was 33.4 ng/mL, and the working range of the determined concentrations was from 42.4 to 1500 ng/mL. Time of the assay-15 min. The developed technique was successfully applied to detect dibutyl phthalate in natural waters with recovery rates from 75 to 115%.

The growing interest in the development of new platforms for the application of Raman spectroscopy techniques in biosensor technologies is driven by the potential of these techniques in identifying chemical compounds, as well as structural and functional features of biomolecules. The effect of Raman scattering is a result of inelastic light scattering processes, which lead to the emission of scattered light with a different frequency associated with molecular vibrations of the identified molecule. Spontaneous Raman scattering is usually weak, resulting in complexities with the separation of weak inelastically scattered light and intense Rayleigh scattering. These limitations have led to the development of various techniques for enhancing Raman scattering, including resonance Raman spectroscopy (RRS) and nonlinear Raman spectroscopy (coherent anti-Stokes Raman spectroscopy and stimulated Raman spectroscopy). Furthermore, the discovery of the phenomenon of enhanced Raman scattering near metallic nanostructures gave impetus to the development of the surface-enhanced Raman spectroscopy (SERS) as well as its combination with resonance Raman spectroscopy and nonlinear Raman spectroscopic techniques. The combination of nonlinear and resonant optical effects with metal substrates or nanoparticles can be used to increase speed, spatial resolution, and signal amplification in Raman spectroscopy, making these techniques promising for the analysis and characterization of biological samples. This review provides the main provisions of the listed Raman techniques and the advantages and limitations present when applied to life sciences research. The recent advances in SERS and SERS-combined techniques are summarized, such as SERRS, SE-CARS, and SE-SRS for bioimaging and the biosensing of molecules, which form the basis for potential future applications of these techniques in biosensor technology. In addition, an overview is given of the main tools for success in the development of biosensors based on Raman spectroscopy techniques, which can be achieved by choosing one or a combination of the following approaches: (i) fabrication of a reproducible SERS substrate, (ii) synthesis of the SERS nanotag, and (iii) implementation of new platforms for on-site testing.

The current COVID-19 pandemic has increased the demand for pathogen detection methods that combine low detection limits with rapid results. Despite the significant progress in methods and devices for nucleic acid amplification, immunochemical methods are still preferred for mass testing without specialized laboratories and highly qualified personnel. The most widely used immunoassays are microplate enzyme-linked immunosorbent assay (ELISA) with photometric detection and lateral flow immunoassay (LFIA) with visual results assessment. However, the disadvantage of ELISA is its considerable duration, and that of LFIA is its low sensitivity. In this study, the modified LFIA of a specific antigen of the causative agent of COVID-19, spike receptor-binding domain, was developed and characterized. This modified LFIA includes the use of gold nanoparticles with immobilized antibodies and 4-mercaptobenzoic acid as surface-enhanced Raman scattering (SERS) nanotag and registration of the nanotag binding by SERS spectrometry. To enhance the sensitivity of LFIA-SERS analysis, we determined the optimal compositions of SERS nanotags and membranes used in LFIA. For benchmark comparison, ELISA and conventional colorimetric LFIA were used with the same immune reagents. The proposed method combines a low detection limit of 0.1 ng/mL (at 0.4 ng/mL for ELISA and 1 ng/mL for qualitative LFIA) with a short assay time equal to 20 min (at 3.5 h for ELISA and 15 min for LFIA). The results obtained demonstrate the promise of using the SERS effects in membrane immuno-analytical systems.

This study provides a comparative assessment of the various nanodispersed markers and related detection techniques used in the immunochromatographic detection of an antibiotic lincomycin (LIN). Improving the sensitivity of the competitive lateral flow immunoassay is important, given the increasing demands for the monitoring of chemical contaminants in food. Gold nanoparticles (AuNPs) and CdSe/ZnS quantum dots (QDs) were used for the development and comparison of three approaches for the lateral flow immunoassay (LFIA) of LIN, namely, colorimetric, fluorescence, and surface-enhanced Raman spectroscopy (SERS)-based LFIAs. It was demonstrated that, for colorimetric and fluorescence analysis, the detection limits were comparable at 0.4 and 0.2 ng/mL, respectively. A SERS-based method allowed achieving the gain of five orders of magnitude in the assay sensitivity (1.4 fg/mL) compared to conventional LFIAs. Therefore, an integration of a SERS reporter into the LFIA is a promising tool for extremely sensitive quantitative detection of target analytes. However, implementation of this time-consuming technique requires expensive equipment and skilled personnel. In contrast, conventional AuNP- and QD-based LFIAs can provide simple, rapid, and inexpensive point-of-care testing for practical use.