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Biosensors based on endonuclease Cas12 provide high specificity in pathogen detection. Sensitive detection using Cas12-based assays can be achieved using trans-cleaved DNA probes attached to simply separated carriers, such as magnetic particles (MPs). The aim of this work was to compare polyA, polyC, and polyT single-stranded (ss) DNA with different lengths (from 10 to 145 nt) as trans-target probes were immobilized on streptavidin-covered MPs. Each ssDNA probe was labeled using fluorescein (5′) and biotin (3′). To compare the probes, we used guide RNAs that were programmed for the recognition of two bacterial pathogens: Dickeya solani (causing blackleg and soft rot) and Erwinia amylovora (causing fire blight). The Cas12 was activated by targeting double-stranded DNA fragments of D. solani or E. amylovora and cleaved the MP–ssDNA conjugates. The considered probes demonstrated basically different dependencies in terms of cleavage efficiency. PolyC was the most effective probe when compared to polyA or polyT probes of the same length. The minimal acceptable length for the cleavage follows the row: polyC < polyT < polyA. The efficiencies of polyC and polyT probes with optimal length were proven for the DNA targets’ detection of D. solani and E. amylovora. The regularities found can be used in Cas12a-based detection of viruses, bacteria, and other DNA/RNA-containing analytes.

Potato blackleg caused by Dickeya spp. bacteria is one of the most important bacterial diseases of potatoes. The rapid spread of this disease in the territory of Russia requires new effective diagnostic tools for the timely detection of infection. To solve this problem, antisera specific to Dickeya spp. were obtained. Polyclonal antibodies isolated from antisera have shown high affinity for the main species of Dickeya spp. ( D. solani, D. dianthicola, D. chrysanthemi, D. dadantii, D. paradisiaca ). Enzyme linked immunosorbent assay (ELISA) and lateral flow immunoassay (LFIA) test systems have been developed based on specific and high affinity antibodies that were obtained. For ELISA, the detection limit was 0.8 105 cells/mL for D. solani and 2 104 cells/mL for D. dianthicola . For LFIA, suitable for use in non-laboratory conditions, the detection limit of D. solani was 2 105 cells/mL and the analysis time was 15 minutes. When testing potato seed material, LFIA test system confirmed positive results of ELISA determination in 75 % of samples, and negative - in 100 % of samples.

A multiplex assay based on recombinase polymerase amplification (RPA) and lateral flow test (LFT) is a desirable tool for many areas. This multiplex assay could be efficiently realized using single-stranded (ss) DNAs located in separate zones on the test strip and bound complementary ssDNA tags of double-stranded (ds) DNA amplicons. Here, we investigate how to enrich multiplex assay capabilities using ssDNAs. Bifunctional oligonucleotide probes integrating (1) a forward primer for RPA, (2) a C9 spacer to stop polymerase, and (3) a ssDNA tag for binding at test strip are developed. The amplicons have a unique individual ssDNA tag at one end and a universal label of fluorescein introducing through a reverse primer at the other end. A conjugate of gold nanoparticles (GNP) with antibodies to fluorescein is used to detect all amplicons. The remainder of primers after RPA interacting with GNP conjugate was found to be a limiting factor for sensitive and specific multiplex assay. The addition of anti-RPA-primers before the use of test strips was proposed to simply and effectively eliminate remaining primers. This approach was successfully applied for the detection of three priority plant RNA viruses: potato virus Y (PVY), -S (PVS) and potato leafroll virus (PLRV). The total time of the assay is 30 min. The multiplex RPA-LFT detected at least 4 ng of PVY per g of plant leaves, 0.04 ng/g for PVS, and 0.04 ng/g for PLRV. The testing of healthy and infected potato samples showed concordance between the developed assay and reverse transcription-polymerase chain reaction. Thus, the capabilities of the proposed universal modules (ssDNA anchors, bifunctional probes, and blocking anti-primers) for multiplex detection of RNA analytes with high specificity and sensitivity were demonstrated.

Recently, immunochromatographic assay (ICA) methods have found a wide range of uses in medical diagnostic, quality control, and consumer safety products because of their rapidity and methodological simplicity of testing; however, ICAs are usually less sensitive than other analytical methods. In this review, we survey modern methodological solutions for ensuring high sensitivity of ICAs based on the use of nanoparticle markers and colorimetric detection and characterize the main direction of such works focusing on lowering the detection limit amplification of the detectable signal generated by a single nanoparticle and forming, during the assay, immune complexes labeled with aggregates consisting of a large number of nanoparticles. The advantages and limitations of different approaches are discussed. We show that their use provides a decrease in the detection limit of ICA by 1–3 orders of magnitude, which makes this method quite competitive compared with instrumental methods of analysis.

CRISPR-Cas12-based biosensors are a promising tool for the detection of nucleic acids. After dsDNA-target-activated Cas12 cleaves the ssDNA probe, a lateral flow test (LFT) is applied for rapid, simple, and out-of-laboratory detection of the cleaved probe. However, most of the existing approaches of LFT detection have disadvantages related to inverted test/control zones in which the assay result depends not only on the cleavage of the probe but also on the second factor: the binding of the non-cleaved probe in the control zone. We proposed a novel platform for the detection of trans-cleaved DNA using a universal DNA-IgG probe and LFT with the sequential direct location of test and control zones. The advantage of the platform consists of the assay result depending only on the cleaved probe. For this, we designed a composite probe that comprise two parts: the DNA part (biotinylated dsDNA connected to ssDNA with fluorescein) (FAM), and the antibody part (mouse anti-FAM IgG). The Cas12, with guide RNA, was activated by the dsDNA-target. The activated Cas12 cleaved the probe, releasing the ssDNA-FAM-IgG reporter that was detected by the LFT. The sandwich LFT was proposed with anti-mouse IgG adsorbed in the test zone and on the surface of gold nanoparticles. We called the platform with direct location zones and direct analyte-signal dependence the DNA-Immunoglobulin Reporter Endonuclease Cleavage Test (DIRECT

The detection limit of lateral flow immunoassay (LFIA) is largely determined by the properties of the label used. We compared four nanoparticle labels differing in their chemical composition and colour: (1) gold nanoparticles (Au NPs), red; (2) Au-core/Pt-shell nanoparticles (Au@Pt NPs), black; (3) latex nanoparticles (LPs), green; and (4) magnetic nanoparticles (MPs), brown. The comparison was carried out using one target analyte—Erwinia amylovora, the causal bacterial agent of fire blight. All nanoparticles were conjugated with antibodies through methods that provide maximum functional coverage like physical adsorption (Au NPs, Au@Pt NPs) and covalent bonding (LPs, MPs). All conjugates demonstrated the same ability to bind with E. amylovora through enzyme-linked immunosorbent assay where optical properties of the nanoparticles do not determine the registered signal. However, half-maximal binding was achieved at different numbers of nanoparticles because they differ in size. All conjugates based on four nanoparticle labels were used for lateral flow assays. As a result, Au@Pt NPs provided the minimal detection limit that corresponded to 103 CFU/mL. Au NPs and LPs detected 104 CFU/mL, and MPs detected 105 CFU/mL. The results highlight that simply choosing a coloured label can significantly affect the detection limit of LFIA.

Verifying the authenticity of food products is essential due to the recent increase in counterfeit meat-containing food products. The existing methods of detection have a number of disadvantages. Therefore, simple, cheap, and sensitive methods for detecting various types of meat are required. In this study, we propose a rapid full-cycle technique to control the chicken or pig adulteration of meat products, including 3 min of crude DNA extraction, 20 min of recombinase polymerase amplification (RPA) at 39 °C, and 10 min of lateral flow assay (LFA) detection. The cytochrome B gene was used in the developed RPA-based test for chicken and pig identification. The selected primers provided specific RPA without DNA nuclease and an additional oligonucleotide probe. As a result, RPA–LFA, based on designed fluorescein- and biotin-labeled primers, detected up to 0.2 pg total DNA per μL, which provided up to 0.001% w/w identification of the target meat component in the composite meat. The RPA–LFA of the chicken and pig meat identification was successfully applied to processed meat products and to meat after heating. The results were confirmed by real-time PCR. Ultimately, the developed analysis is specific and enables the detection of pork and chicken impurities with high accuracy in raw and processed meat mixtures. The proposed rapid full-cycle technique could be adopted for the authentication of other meat products.

The combination of recombinase polymerase amplification (RPA) and lateral flow test (LFT) is a strong diagnostic tool for rapid pathogen detection in resource-limited conditions. Here, we compared two methods generating labeled RPA amplicons following their detection by LFT: (1) the basic one with primers modified with different tags at the terminals and (2) the nuclease-dependent one with the primers and labeled oligonucleotide probe for nuclease digestion that was recommended for the high specificity of the assay. Using both methods, we developed an RPA-LFT assay for the detection of worldwide distributed phytopathogen—alfalfa mosaic virus (AMV). A forward primer modified with fluorescein and a reverse primer with biotin and fluorescein-labeled oligonucleotide probe were designed and verified by RPA. Both labeling approaches and their related assays were characterized using the in vitro-transcribed mRNA of AMV and reverse transcription reaction. The results demonstrated that the RPA-LFT assay based on primers-labeling detected 103 copies of RNA in reaction during 30 min and had a half-maximal binding concentration 22 times lower than probe-dependent RPA-LFT. The developed RPA-LFT was successfully applied for the detection of AMV-infected plants. The results can be the main reason for choosing simple labeling with primers for RPA-LFT for the detection of other pathogens.

Magnetic beads (MBs) are often considered as an effective carrier in heterogeneous assays due to the simplicity of separation and washing, and the ability to increase and control the surface area. However, the effect of the MBs surface on the analytical parameters is poorly characterized and is often postulated from intuitive considerations. Herein, experimental evaluation through the comparison of MBs and microwell plate was carried out using the miRNA-141 (biomarker for cancer) as a target, the detection of which was performed by chemiluminescent assay with a homogeneous mismatched catalytic hairpin assembly (mCHA) reaction. The mCHA reaction produced double-stranded (ds) DNA labeled at one end with fluorescein (Flu) for capture with anti-Flu antibodies immobilized on a solid carrier, on the other end with biotin for recognition by streptavidin-polyperoxidase conjugate. The conditions of immobilization of anti-Flu antibody on MBs (a diameter of 440 nm) performed using a carbodiimide method were optimized by varying the antibody concentration in the reaction solution. It was shown that the dependence of chemiluminescent signal as a function of the concentration of anti-FluAb-MBs conjugates had a bell-shaped character. The maximum chemiluminescence was produced at the concentration of the conjugates of 2 × 10

A method is proposed for reducing the detection limit of enzyme-linked immunosorbent assay (ELISA) of potato virus X, based on the multiple introduction of tyramine into immune complexes and the subsequent detection of an enzyme label. During ELISA, a step by step formation of sandwich complex containing immobilized antibodies – potato virus X – horseradish peroxidase conjugate with antibodies to the virus was carried out. Peroxidase catalyzed the multiple insertion of a tyramine-biotin label into protein molecules, providing signal amplification upon the addition of the streptavidin-polyperoxidase conjugate. The conditions of the assay that ensure a high degree of amplification and a minimum background signal were established. The use of tyramine amplification made it possible to lower the detection limit by more than 30 times (from 100 to 3 ng/ml) when assayed in the buffer and extracts of potato leaves, slightly increasing its duration. Tyramine amplification is based on the use of universal reagents and can be used to reduce the detection limit of ELISA for other antigens.

The use of gold nanoparticles (GNPs) as carriers for the decrease in the detection limit of a fluorescence polarization (FP) aptamer assay is proposed. The common FP assay is based on the use of polarized exciting light and changes in the polarization of emitted light by the fluorophore–analyte conjugate before and after its binding with a receptor of the target analyte. Aptamers’ application as receptors in such an assay is limited due to their low molecular weight and, consequently, insufficient influence on polarization of emitted light. This limitation can be overcome by the inclusion of aptamers in larger intermolecular complexes. In the present work, the advantages of GNPs as unified, stable, and simply modified carriers for aptamers are demonstrated. The FP aptamer assay is realized with the use of GNPs with average diameter of 8.7 nm and ochratoxin A (OTA) as the target analyte. Finally, the assay is tested for OTA control in spiked white wine. The reached limit of detection is 2.3 µg/kg, which is 25-fold lower as compared to native aptamer. The time of the assay is 15 min. The universality of the proposed approach makes it possible to use aptamers for FP assays of various low-molecular-weight substances.

Cadmium (Cd), lead (Pb) and mercury (Hg) presence was investigated in the muscle tissue of gilthead seabream and seabass, collected from various aquaculture sites of the Aegean and Cretan Sea as well as from the fish market (fisheries). Risk for the Greek population through consumption of these species was estimated using two approaches: Target Hazard Quotient (THQ) and Hazard Index (HI). All heavy metal levels in the fish tissue were below the established safe limits for consumption. Metal accumulation was found to differ amongst mode of production, species, location and seasonality. Seabass demonstrated higher Hg and lower Cd concentrations than seabream, Hg and Pb seem to be more accumulated in closed seas and Pb values displayed a linear increasing trend from warmer to colder periods. Regression analysis revealed that the main contributing factor to Cd accumulation is species (beta: -0.28, 95%CI: -0.48 to -0.09); lead is predominately affected by seasonality (beta: 0.44, 95%CI: 0.29 to 0.59), Hg accumulation is mainly affected by location (beta: -0.32, 95%CI: -0.61 to -0.03) while wild seabream accumulates greater levels for Hg and Pb than farmed. Risk analysis demonstrated that consumption of the studied species, is safe for all metals (HI 0.460 and TTHQ 0.299).

The experimental data on the oral toxicity of nanostructured amorphous silica (SiO2), widely used in food supplements, pharmaceuticals, and cosmetics, in terms of its in vivo effect on the immune system, are contradictory. Therefore, this study aimed to assess the rat&rsquo, s immune function after SiO2 oral administration. In the first experiment, SiO2 was daily orally administered to Wistar rats for 92 days in doses of 0.1, 1.0, 10, and 100 mg/kg of body weight (bw). In the second 28-day experiment, SiO2 in a dose of 100 mg/kg bw was daily orally administered to rats parenterally immunized with the food allergen ovalbumin (OVA) for the reproduction of systemic anaphylaxis reaction. Together with integral indices, we assessed intestinal permeability to protein macromolecules, hematology, CD45RA+, CD3+, CD4+, CD8+, and CD161a+ cells, cytokines TNF-&alpha, IL-6, and IL-10, and IgG to OVA. The results obtained showed that SiO2 has no effect on the severity of the anaphylactic reaction, but is capable inducing a toxic effect on the T-cell immune systems of rats. Estimated no observed adverse effect level NOAEL for SiO2 ranges up to 100 mg/kg bw in terms of its daily consumption for 1&ndash, 3 months. Using SiO2 as a food additive should be the subject of regulation.

An assay was developed to detect the potato spindle tuber viroid (PSTVd), a dangerous plant pathogen that causes crop damage resulting in economic losses in the potato agriculture sector. The assay was based on the reverse transcription and recombinase polymerase amplification (RT-RPA) of PSTVd RNA coupled with amplicon detection via lateral flow assay (LFA). Primers labeled with fluorescein and biotin were designed for RT-RPA for effective recognition of the loop regions in the high-structured circular RNA of PSTVd. The labeled DNA amplicon was detected using lateral flow test strips consisting of a conjugate of gold nanoparticles with antibodies specific to fluorescein and streptavidin in the test zone. The RT-RPA-LFA detected 106 copies of in vitro transcribed PSTVd RNA in reaction or up to 1:107 diluted extracts of infected plant leaves. The assay took 30 min, including the RT-RPA stage and the LFA stage. The testing of healthy and infected potato samples showed full concordance between the developed RT-RPA-LFA and quantitative reverse transcription polymerase chain reaction (RT-qPCR) and the commercial kit. The obtained results proved the feasibility of using the developed assay to detect PSTVd from a natural source.

We report the approach for the detection of Au@Pt core@shell nanoparticles (nanozymes) with peroxidase-mimicking activity (PMA) in samples with high endogenous peroxidase activity (EPA). Unlike the endogenous peroxidases in plant extracts that are inhibited by elevated H2O2 (>20 mM), the PMA of nanozymes was stable in concentrated H2O2 (up to 4 M). Such a different stability of enzymes and Au@Pt to the substrate allowed for eliminating EPA and detecting only nanozymes. The developed approach was used for reaching a lower limit of detection (LOD) and eliminating the background for the lateral flow immunoassay (LFIA) of the important plant pathogen potato virus X (PVX) in leaf and tuber extracts. Using the PMA of Au@Pt, the LOD was reduced to 4 and 8 pg/mL in tuber and leaf extracts, respectively. The LOD values are 250- and 500-times lower in comparison with LFIA with conventional gold nanoparticles. The developed approach of peroxidases inhibition is universal for bioanalytical methods, and its applicability was confirmed by the elimination of EPA in three matrixes (serum, potato leaf and tuber extracts).

Conjugates of gold nanoparticles (GNPs) and antibodies are widely used in various fields of biochemistry and microbiology. However, the procedure for obtaining such conjugates remains precarious, and the properties of conjugates differ significantly for different antibody clones. One of the most common problems is the aggregation of GNPs in the course of their conjugation with antibodies. This article considers an example of the conjugation of monoclonal antibodies with non-stable aggregating product. The composition of the antibody preparation was studied using electrophoresis, asymmetrical flow field-flow fractionation, and ultracentrifugation. It was shown that the component that causes the aggregation of the GNPs is the light chains of immunoglobulins that appear due to the spontaneous decay of the antibodies. After separation of the fraction with a molecular weight of less than 30 kDa, stable conjugates of antibodies with GNPs were obtained. The high functional activity of the obtained conjugates was confirmed by immunochromatography.

The fluorescent properties of ligands can change when they bind to specific receptors. Modulated by the transition of the ligand from the free to the bound state, fluorescence makes it possible both to detect this ligand and quantitatively register its binding. We characterized the interaction of ochratoxin A (OTA) with the specific G-quadruplex aptamer through excitation-emission matrix fluorescence spectroscopy. It was shown that the formation of the complex changes the OTA fluorescence spectrum both in the region of the main peak at λex/λem 380/430 nm and in the region of peak at λex/λem 265/425 nm. At pH 8.5 and OTA concentration of 30 nM, this peak is smaller in intensity than the main peak of fluorescence. The formation of the complex with the aptamer leads to an increase of the fluorescence at λex/λem 265/425 nm up to 6.5 times, which makes it up to 4.9 times more intense than fluorescence at 380/430 nm. Fluorescence of the G-quadruplex aptamer (donor) takes part in increasing of the OTA (acceptor) emission at λex/λem 265/425 nm due to the resonance energy transfer. The concentration regularities of the modulated fluorescence of OTA at λex/λem 265/425 nm have been studied. Their correspondence to the calculations of complexation conducted on the basis of the dissociation constant is shown.

Registration of fluorescence anisotropy (FA) allows for characterizing the interactions of ligands with aptamers and other receptors under homogeneous conditions without reagent immobilization, prolonged incubations, and product separation. We proposed an approach for aptamer affinity determination by FA taking into account the difference in label fluorescence before and after complexation. The detailed step by step scheme using a native and fluorescently labeled ligand was described and justified in the paper. The scheme ensures the exclusion of data with low reliability and establishes valid criteria for selecting optimal concentrations of reagents (labeled ligand and aptamer) used in the experiments. The approach was experimentally tested using ochratoxin A (OTA), its fluorescein-labeled derivative (OTA-Flu), and the aptamer binding them. We demonstrated that it allows minimizing the influence of fluorescence change to accurately determine the dissociation constant. On the basis of FA registration, the...

This study presents the joint use of magnetic nanoparticles (MNPs) and gold nanoparticles (GNPs) for double enhancement in a lateral flow immunoassay (LFIA). The study realizes two types of enhancement: (1) increasing the concentration of analytes in the samples using conjugates of MNPs with specific antibodies and (2) increasing the visibility of the label through MNP aggregation caused by GNPs. The proposed strategy was implemented using a LFIA for potato virus X (PVX), a significant potato pathogen. MNPs conjugated with biotinylated antibodies specific to PVX and GNPs conjugated with streptavidin were synthesized and characterized. The LFIAs with and without the proposed enhancements were compared. The double-enhanced LFIA achieved the highest sensitivity, equal to 0.25 ng mL-1 and 32 times more sensitivity than the non-enhanced LFIA (detection limit: 8 ng mL-1). LFIAs using one of the types of amplification (magnetic concentration without GNPs-causing aggregation or MNP aggregation without the concentration stage) showed intermediate levels of sensitivity. The double-enhanced LFIA was successfully used for PVX detection in potato leaves. The results for PVX detection in the infected plants were similar for the double-enhanced LFIA developed and the conventional LFIA based on the GNP conjugates; however, the new system provided significant coloring enhancement. This study confirmed that a simple combination of MNPs and GNPs has great potential for high-sensitivity detection and could possibly be adopted for LFIAs of other compounds.

Rapid, sensitive, and timely diagnostics are essential for protecting plants from pathogens. Commonly, PCR techniques are used in laboratories for highly sensitive detection of DNA/RNA from viral, viroid, bacterial, and fungal pathogens of plants. However, using PCR-based methods for in-field diagnostics is a challenge and sometimes nearly impossible. With the advent of isothermal amplification methods, which provide amplification of nucleic acids at a certain temperature and do not require thermocyclic equipment, going beyond the laboratory has become a reality for molecular diagnostics. The amplification stage ceases to be limited by time and instruments. Challenges to solve involve finding suitable approaches for rapid and user-friendly plant preparation and detection of amplicons after amplification. Here, we summarize approaches for in-field diagnostics of phytopathogens based on different types of isothermal amplification and discuss their advantages and disadvantages. In this review, we consider a combination of isothermal amplification methods with extraction and detection methods compatible with in-field phytodiagnostics. Molecular diagnostics in out-of-lab conditions are of particular importance for protecting against viral, bacterial, and fungal phytopathogens in order to quickly prevent and control the spread of disease. We believe that the development of rapid, sensitive, and equipment-free nucleic acid detection methods is the future of phytodiagnostics, and its benefits are already visible.

Rapid non-laboratory screening of plants for pathogenic viruses crucially influences crop yields in modern agricultural technologies. The aim of this study was to develop a highly-sensitive lateral flow immunoassay (LFIA) for rapid detection of potato leafroll virus (PLRV), an infectious agent of one of the most widespread potato diseases. The proposed LFIA combines the formation of sandwich immune complexes with gold nanoparticles (GNP) as labels and silver enhancement. The enhancement stage was realized using mixture of silver lactate and hydroquinone and subsequent addition of chloride-containing buffer to stop silver reduction. LFIA with silver enhancement was 15 times more sensitive (detection limit 0.2 ng/mL; 15 min) compared with conventional LFIA (detection limit 3 ng/mL; 10 min). The enhanced LFIA detected PLRV in leaves��� extracts of infected potato in dilutions higher than enzyme-linked immunosorbent assay.

This comprehensive study is related to gold nanoparticles (GNPs) conjugated with antibodies. The goal of the study is to determine the minimal concentration of antibodies for conjugate synthesis when the conjugates have high antigen-capturing activity. Two systems were studied: gold nanoparticles conjugated with monoclonal antibodies (mAb-GNP) specific to Helicobacter pylori and gold nanoparticles conjugated with polyclonal antibodies (pAb-GNP) specific to mouse immunoglobulins. Several conjugates were synthesized with different GNP-to-antibody molar ratios (from 1:1 to 1:245) through nondirectional and noncovalent immobilization on a surface of GNPs with a diameter of 25.3 ± 4.6 nm. The maximal antigen-capturing activities and equilibrium constants of the conjugates correlate with the formation of a constant hydrodynamic radius of the conjugates for mAb-GNP (GNP to antibody molar ratio 1:58) and with the stabilizing concentration by flocculation curves for pAb-GNP (GNP to antibody molar ratio 1:116). The application of the conjugates to the lateral flow immunoassay shows that the antibody concentrations used for the conjugation can be reduced (below the stabilizing concentration) without losing activity for the mAb-GNP conjugates. The findings highlight that the optimal concentration of antibodies immobilized on the surface of GNPs is not always equal to the stabilizing concentration determined by the flocculation curve.

The influence of Au@Pt nanoparticles’ composition, morphology, and peroxidase-mimicking activity on the limit of detection (LOD) of lateral flow immunoassay (LFIA) has been investigated. Fourteen types of nanoparticles were synthesized by varying the concentration of Pt4+ (20–2000 μM), using gold nanoparticles (GNP, diameter 20.0 ± 2.6 nm) as the seeds and ascorbic acid as a reducing agent. Au@Pt nanoparticles and GNPs were conjugated with antibodies specific to the target analyte, a widespread and dangerous phytopathogenic bacteria species (Clavibacter michiganensis). We found that the 100-fold growth of the Pt4+ concentration was accompanied by an increase of the Au@Pt nanoparticle diameter (24–55 nm) and surface area with the formation of urchin-shaped morphology. These changes led to a 70-fold increase in peroxidase-mimicking activity in the solution (specific activity 0.06–4.4 U mg−1) and a 30-fold decrease in LOD using the catalytic activity of Au@Pt. The Au@Pt nanoparticles synthesized at 1000–2000 μM of Pt4+ demonstrated statistically indistinguishable catalytic activity. The highest sensitivity of LFIA was reached for Au@Pt nanoparticles synthesized at Pt4+ concentration equal to 1000 μM. Au@Pt nanoparticles saved most of their peroxidase-mimicking activity, whereas endogenous plant peroxidases were completely inhibited by sodium azide. The LOD of LFIA with Au@Pt nanoparticles synthesized at 1200 μM of Pt4+ was 300 colony-forming units (CFU) per mL of buffer and 500 CFU per mL of potato tuber extract, which provides 330- and 200-fold improvement compared to the conventional LFIA with GNPs. The assay consists of three rapid 5-min stages, namely, extraction, lateral flow, and color enhancement (oxidation of diaminobenzidine by Au@Pt nanoparticles). LFIA with the urchin Au@Pt nanoparticles allows the detection of latent bacterial infections rapidly without equipment or special skills.

We propose nucleic acid lateral flow assay (LFA) coupled with reverse transcription recombinase polymerase amplification (RT-RPA) resulting from step-by-step multiparametric adjustments to both RT-RPA reactions and LFA interactions. The assay was realized for RNA virus detection using the example of potato virus X (PVX), a dangerous phytopathogen. The assay stages were adjusted for sensitive detection. (1) DNA target was designed and verified. A fragment (146 bp) of coat protein gene (gp5) and biotin-/fluorescein-labeled forward/reverse primers were chosen to produce target amplicons. (2) In a test strip, the construction advantage of the realization of the highest affinity interaction (biotin–streptavidin in our research) through gold nanoparticle conjugate (streptavidin immobilized on the GNP surface) was demonstrated. (3) RPA with reverse transcription was adjusted including primer concentration, order of components’ mixing, and reaction temperature. Due to the adjustments, the assay was able to detect 0.14 ng PVX per g potato leaves at 30 min. The PVX assay was 260 times more sensitive than conventional lateral flow assay based on antibodies and demonstrated the same sensitivity as PCR detection. The proposed adjustments are applicable for ultrasensitive and rapid detection of various RNA viruses.

The combination of isothermal nucleic acid amplification and lateral flow assay (LFA) provides highly sensitive non-laboratory (“point-of-care”) detection. The aim of this study is to investigate the recognition on lateral flow membranes of DNA targets with different lengths as products of recombinase polymerase amplification (RPA). We produced double-stranded DNA with lengths of 50, 100, 150, 200, and 300 bp. Each DNA target was functionalized with biotin and fluorescein (FAM). Kinetic and equilibrium constants of the interaction of FAM at the 5′-end of DNA with anti-FAM antibodies did not depend on DNA length. Gold nanoparticles (GNPs) with diameters of 17.4 ± 1.0 nm were conjugated with anti-FAM antibodies and streptavidin. LFA was performed in two schemes: 1) anti-FAM antibodies immobilized in the test zone, GNP–streptavidin conjugates recognized as DNA; 2) streptavidin immobilized in the test zone, GNP‒anti-FAM antibodies conjugates recognized as DNA. Considering that the components of the RPA mixture caused the aggregation of the GNP–streptavidin conjugate in contradistinction to conjugate with anti-FAM antibodies, we found that 150 bp was the most promising length for the DNA target. For this length, a detection limit was achieved up to 70 pM that was approximately 10 times lower than for 50-bp DNA in the same scheme. Moreover, we showed that high concentrations of primers containing FAM or biotin competed with the DNA target on lateral flow membranes. These results demonstrated that a DNA length should be considered when designing RPA–LFA systems to detect DNA targets with high sensitivity.

The authors describe a method for the improvement of the sensitivity of immunoassays. This was achieved by a combination of immunoassay and an amplification based on the use of a multiplied DNA probe. The immunoassay was enhanced via recombinant polymerase amplification (RPA). It enables fast (35 min) isothermal multiplication of DNA at 37 °C. This concept was demonstrated for a sandwich immunoassay that is making use of magnetic nanoparticles conjugated to first specific antibodies and then to second specific antibodies linked to reporter DNA via biotin-streptavidin binding. Reporter DNA multiplied by RPA was quantified fluorometrically via the carboxyfluorescein label. Human cardiac troponin T (cTnT), a biomarker for acute myocardial infarction, was used as the target of the immunoRPA (iRPA). The assay can detect cTnT in serum and in plasma within 2 h, with detection limits as low as 12.5 ± 1.1 pg•mL−1 and 9.4 ± 2.1 pg•mL−1, respectively. This represents increased sensitivity and decreased assay time compared to classical ELISAs (3.5 ± 0.3 ng•mL−1) or immuno-PCR (4.3 ± 0.8 ng•mL−1) that were carried out using the same immunoreagents and reporter DNA. The good performance of this iRPA was underlined by its successful application to the analysis of plasma samples. The iRPA has significant advantages relative to other DNA amplification-based immunoassays due to isothermal conditions and analysis at 37 °C.

The authors describe a method for the improvement of the sensitivity of immunoassays. This was achieved by a combination of immunoassay and an amplification based on the use of a multiplied DNA probe. The immunoassay was enhanced via recombinant polymerase amplification (RPA). It enables fast (35 min) isothermal multiplication of DNA at 37 °C. This concept was demonstrated for a sandwich immunoassay that is making use of magnetic nanoparticles conjugated to first specific antibodies and then to second specific antibodies linked to reporter DNA via biotin-streptavidin binding. Reporter DNA multiplied by RPA was quantified fluorometrically via the carboxyfluorescein label. Human cardiac troponin T (cTnT), a biomarker for acute myocardial infarction, was used as the target of the immunoRPA (iRPA). The assay can detect cTnT in serum and in plasma within 2 h, with detection limits as low as 12.5 ± 1.1 pg•mL

Erwinia amylovora causes a quarantine disease called fire blight that affects most plants from the Rosaceae family. An efficient and rapid disease diagnosis tool is needed to prevent the spread of the pathogen. The main objective of this study was to develop a lateral flow immunoassay (LFIA) to detect E. amylovora and compare different organs of plants for optimization of LFIA testing. A total of 11 strains of E. amylovora and related species were tested for specificity of the developed LFIA. The detection limit of E. amylovora was equal to 4 × 105 CFU mL−1 in plant extracts. LFIA showed high specificity and did not demonstrate positive results with non-related species. Meanwhile, LFIA's effectiveness was confirmed through testing artificially infected leaf samples of apple, pear, and black raspberry. Reliable results were obtained 10 min after the start of LFIA for all testing strains. Different plant organs (121 samples) comprising apple, pear, hawthorn, quince, blackthorn, and cherry from naturally infected areas with symptoms of varying severity were tested. The LFIA was performed using samples from leaves, twigs, flowers, fruitlets, and bacterial ooze; for confirmation commercial kits based on fluorescent amplification–based specific hybridization PCR were used. Using several samples from one plant (cluster) significantly increased the accuracy of infected plant detection, the overlap of LFIA and PCR were equal to 70.2% for individual samples and 93.5% for clusters. Observed recovery of E. amylovora for different organs differed by up to 20%. We found out that using vascular tissues was better than using leaf extracts. This result demonstrates that LFIA's effectiveness improved when more appropriate samples were used.

Here we report on the unexpected electrophoretic behavior of complexes between rod-like virus particles (virions) and bivalent antibodies. The multiple complexes formed by the virions and antibodies migrated with electrophoretic mobilities of much greater absolute values than those of the unbound virions or antibodies while typically complexes have mobilities intermediate to those of their components. We hypothesized that the mobilities of unusually high absolute values are caused by the cross-linking of virions by bivalent antibodies into aggregates with prominent side-to-side binding. Theoretically, the mobility of such aggregates should be proportional to the square root of the number of cross-linked virions. The formation of virion aggregates with prominent side-to-side binding was confirmed by atomic force microscopy. The dependence of the aggregate mobility on the number of cross-linked virions can be used to estimate this number.