Irina V. Safenkova

The CRISPR/Cas12a system is widely used in nucleic-acid diagnostics because Cas12a couples guide-directed recognition of a cis -target with indiscriminate collateral trans -cleavage of ssDNA. Controlling these activities through DNA structure can tune diagnostic signals. G-quadruplexes (G4s) are strong structural regulators, yet their ability to modulate Cas12a activation and collateral cleavage remains undefined. Here we focused on the G4 scaffold (TGGG)n and tested it with LbCas12a and AsCas12a by real-time cleavage assays, circular dichroism, FRET and denaturing PAGE. In the cis position, compact (TGGG)n G4s activated Cas12a; for the most stable (TGGG) ₅ , k _cat /K _M was 5.9×10 ⁴ and 8.9×10 ⁴ M ⁻¹ s ⁻¹ for LbCas12a and AsCas12a, respectively. K ⁺ reduced cis -activation rates by approximately 10-fold, depending on scaffold and temperature. FRET and denaturing PAGE showed that productive cis -recognition involves G4 unfolding followed by target-strand cleavage. In the trans position, compact G4s fully resisted collateral reporter cleavage. Core disruption and G-rich non-G4 controls restored reporter cleavage, showing that resistance depends on G4 architecture rather than guanine content. These data define a compact G4 scaffold that remains functionally trans -resistant while retaining guide-dependent cis -target competence. Thus, compact G4 folding provides a programmable structural mechanism for separating Cas12a activation from reporter cleavage, opening a route to signal-gated diagnostic designs. Graphical

CRISPR/Cas12a systems coupled with lateral flow tests (LFTs) are a promising route to rapid, instrument-free nucleic acid diagnostics due to conversion target recognition into a simple visual readout via cleavage of dual-labeled single-stranded DNA reporters. However, the conventional CRISPR/Cas12a–LFT system is constructed in a format where the intact reporter should block nanoparticle conjugate migration and can produce false-positive signals and shows strong dependence on component stoichiometry and kinetics. Here, we present the first combined experimental and theoretical analysis quantifying these limitations and defining practical solutions. The experimental evaluation included 480 variants of LFT configuration with reporters differing in the concentration of interacting components and the kinetic conditions of the interactions. The most influential factor leading to 100% false-positive results was insufficient interaction time between the components; pre-incubation of the conjugate with the reporter for 5 min eliminated these artifacts. Theoretical analysis of the LFT kinetics based on a mathematical model confirmed kinetic constraints at interaction times below a few minutes, which affect the detectable signal. Reporter concentration and conjugate architecture represented the second major factors: lowering reporter concentration to 20 nM and using smaller gold nanoparticles with multivalent fluorescent reporters markedly improved sensitivity. The difference in sensitivity between various LFT configurations exceeded 50-fold. The combination of identified strategies eliminated false-positive reactions and enabled the detection of up to 20 pM of DNA target (the hisZ gene of Erwinia amylovora, a bacterial phytopathogen). The strategies reported here are general and readily transferable to other DNA targets and CRISPR/Cas12a amplification-free diagnostics.

CRISPR/Cas12a-based assays, when integrated with lateral flow tests (LFTs), provide highly specific nucleic acid detection in a simple, rapid, and equipment-free format. Nevertheless, traditional DNA probes utilized for cleavage by Cas12a have limitations as the cleaved probe only has one label. To overcome this challenge, we engineered a novel type of DNA probe with multiple fluorescein (FAM) labels and a biotin-labeled single-stranded DNA fragment (polyFAM probe). The cleaved polyFAM parts of the probes were detected using a specially designed sandwich LFT, where FAM-specific antibodies were immobilized in the test zone and conjugated with gold nanoparticles. The LFT ensured accurate recognition of the cleaved polyFAM fragments within 10 min. A comparison of five distinct polyFAM probes revealed that the highest signal-to-noise ratio was achieved with a tripod-branched probe synthesized via trebler phosphoramidite modification. Each arm of the tripod probe consists of a hexaethylene glycol spacer ending in a FAM label. Upon Cas12a cleavage, the tripod structure carrying three FAMs is released and detected by LFT. A rapid magnetic separation strategy was subsequently implemented, facilitating the efficient removal of uncleaved probes via biotin–streptavidin capture within 5 min. The CRISPR/Cas12a–tripod–LFT strategy demonstrated excellent sensitivity without preamplification, with a detection Limit of 1.4 pM for DNA target of Salmonella Typhimurium. The CRISPR/Cas12a-tripod-LFT with preliminary loop-mediated isothermal amplification enabled the detection of as few as 0.3 cells per reaction. This innovative tripod probe with corresponding LFT creates a universal, sensitive, rapid, and equipment-free biosensing platform for CRISPR/Cas12a-based diagnostics in point-of-care applications. Graphical Abstract

CRISPR/Cas12a-based assays, when integrated with lateral flow tests (LFTs), provide highly specific nucleic acid detection in a simple, rapid, and equipment-free format. Nevertheless, traditional DNA probes utilized for cleavage by Cas12a have notable limitations as the cleaved probe only has one label. To overcome this challenge, we engineered a novel type of DNA probe with multiple fluorescein (FAM) labels and a biotin-labeled single-stranded DNA fragment (polyFAM probe). The cleaved polyFAM parts of probes were detected using a specially designed sandwich LFT, where FAM-specific antibodies were immobilized in the test zone and conjugated with gold nanoparticles. The LFT ensured accurate recognition of the cleaved polyFAM fragments within 10 minutes. A comparison of five distinct polyFAM probes revealed that the highest signal-to-noise ratio was achieved with a tripod-branched probe synthesized via trebler phosphoramidite modification. Each arm of the tripod probe consists of a hexaethylene glycol spacer ending in a FAM label. Upon Cas12a cleavage, the tripod structure carrying three FAMs is released and detected by LFT. A rapid magnetic separation strategy was subsequently implemented, facilitating the efficient removal of uncleaved probes via biotin–streptavidin capture within 5 minutes. The CRISPR/Cas12a–tripod–LFT strategy demonstrated excellent sensitivity without preamplification, with a detection limit of 1.4 pM for DNA target of Salmonella Typhimurium. The CRISPR/Cas12a-tripod-LFT with preliminary loop-mediated isothermal amplification enabled the detection of as few as 0.3 cells per reaction. This innovative tripod probe with corresponding LFT creates a universal, sensitive, rapid, and equipment-free biosensing platform for CRISPR/Cas12a-based diagnostics in point-of-care applications.

In the modern food production technologies, the tools and means of simple and rapid testing raw materials, intermediate products and the final ready-to-consume food products are in high demand. This monitoring allows determining the content of toxic and pathogenic contaminants and confirms the compliance of the objects being tested with the established regulatory requirements. Mobile tests tools and means (so called test systems) provide the opportunity of wide range monitoring without involving the specialized laboratories and highly qualified specialists. Thus, test systems for detection of toxic and pathogenic contaminants serve as the useful addition to confirming instrumental analytical methods. An actively developing approach for this field testing is the using of immunochromatographic test strips, in which strips all the necessary reagents are applied to the membrane components of the analytical system. Contact of the test strip with the sample being tested, initiates all further interactions and generates the recordable or visually assessable optical signal. The market of test systems based on immunochromatographic analysis is constantly growing, thus offering the permanently widening choice of solutions. However, in recent years there has been a real boom of new developments in immunochromatography field, thus offering various options for highly sensitive and information capacitive analytical systems. This study systematizes these developments and provides their comparative assessment in terms of prospects for their technological implementation and practical application in the coming years. The opportunities of designing the antibodies and alternative receptor molecules for controlling the affinity and the selectivity of recognition of the compounds being monitored are considered. The advantages and limitations of the new nanodispersed markers and non-optical methods for their registration in immunochromatography are discussed. The methods for quantitative assessment of the contaminants content via immunochromatography are characterized. The developed design options of the test systems for multiplex control — simultaneous detection of several compounds — are presented. Examples of integration of immunochromatographic tests with the systems of automatic registration, processing, transfer, storage and analysis of results of numerous tests are represented.

To ensure the safety of foodstuffs, widespread non-laboratory monitoring for pathogenic contaminants is in demand. A suitable technique for this purpose is lateral flow immunoassay (LFIA) which combines simplicity, rapidity, and productivity with specific immune detection. This study considered three developed formats of LFIA for Salmonella Typhimurium, a priority pathogenic contaminant of milk. Common sandwich LFIA with all immunoreagents pre-applied to the test strip (format A) was compared with incubation of the sample and (gold nanoparticle—antibody) conjugate, preceding the lateral flow processes (format B), and sequential passages of the sample and the conjugate along the test strip (format C). Under the chosen conditions, the detection limits and the assay times were 3 × 10⁴, 1 × 10⁵, and 3 × 10⁵ cells/mL, 10, 15, and 20 min for formats A, B, and C, respectively. The selected format A of LFIA was successfully applied to test milk samples. The sample’s dilution to a fat content of 1.0% causes pathogen detection, with 70–110% revealing and 1.5–8.5% accuracy. The obtained results demonstrate that the developed LFIA allows the detection of lower concentrations of Salmonella cells and, in this way, accelerates decision-making in food safety control.

Pectobacterium brasiliense is a bacterial phytopathogen that causes soft and black rot and actively spreads worldwide. Our study is the first development of immunoassays for detecting P. brasiliense. We immunized rabbits and obtained serum with an extremely high titer (1:10⁸). Isolated polyclonal antibodies were tested by enzyme-linked immunosorbent assay (ELISA) using 18 closely related strains and 5 non-related bacterial pathogens. No cross-reactivity was found concerning the tested pathogens. The ELISA of P. brasiliense was developed in a double-antibody sandwich format with a detection limit of 1.5 × 10⁴ cells/mL. A lateral flow immunoassay (LFIA) for P. brasiliense was also developed in a double-antibody sandwich format with a detection limit of 1 × 10⁵ cells/mL. The results of P. brasiliense cells testing with LFIA in plant matrix showed a high correlation (R² = 0.932) between concentrations of added and revealed cells. When testing potato seed material, ELISA and LFIA confirmed 75 and 66% of positive samples according to real-time PCR, respectively. For negative samples, ELISA showed 84% coincidence, and LFIA coincided with PCR for 89% of samples. Thus, the developed immunoassays can be used to evaluate plant material in poorly equipped conditions or under field testing.