Engineering of DNA Structures Attached to Magnetic Particles for Effective Trans- and Cis-Cleavage in Cas12-Based Biosensors.

Sequence-specific endonuclease Cas12-based biosensors have rapidly evolved as a strong tool to detect nucleic acids. Magnetic particles (MPs) with attached DNA structures could be used as a universal platform to manipulate the DNA-cleavage activity of Cas12. Here, we propose nanostructures of trans- and cis-DNA targets immobilized on the MPs. The main advantage of the nanostructures is a rigid double-stranded DNA adaptor that distances the cleavage site from the MP surface to ensure maximum Cas12 activity. Adaptors with different lengths were compared by detecting the cleavage by fluorescence and gel electrophoresis of the released DNA fragments. The length-dependent effects for cleavage on the MPs' surface were found both for cis- and trans-targets. For trans-DNA targets with a cleavable 15-dT tail, the results showed that the optimal range of the adaptor length was 120-300 bp. For cis-targets, we varied the length and location of the adaptor (at the PAM or spacer ends) to estimate the effect of the MP's surface on the PAM-recognition process or R-loop formation. The sequential arrangement of an adaptor, PAM, and a spacer was preferred and required the minimum adaptor length of 3 bp. Thus, with cis-cleavage, the cleavage site can be located closer to the surface of the MPs than with trans-cleavage. The findings provide solutions for efficient Cas12-based biosensors using surface-attached DNA structures.

Modulation of Aptamer-Ligand-Binding by Complementary Oligonucleotides: A G-Quadruplex Anti-Ochratoxin A Aptamer Case Study.

Short oligonucleotides are widely used for the construction of aptamer-based sensors and logical bioelements to modulate aptamer-ligand binding. However, relationships between the parameters (length, location of the complementary region) of oligonucleotides and their influence on aptamer-ligand interactions remain unclear. Here, we addressed this task by comparing the effects of short complementary oligonucleotides (ssDNAs) on the structure and ligand-binding ability of an aptamer and identifying ssDNAs' features that determine these effects. Within this, the interactions between the OTA-specific G-quadruplex aptamer 1.12.2 (5'-GATCGGGTGTGGGTGGCGTAAAGGGA GCATCGGACA-3') and 21 single-stranded DNA (ssDNA) oligonucleotides complementary to different regions of the aptamer were studied. Two sets of aptamer-ssDNA dissociation constants were obtained in the absence and in the presence of OTA by isothermal calorimetry and fluorescence anisotropy, respectively. In both sets, the binding constants depend on the number of hydrogen bonds formed in the aptamer-ssDNA complex. The ssDNAs' having more than 23 hydrogen bonds with the aptamer have a lower aptamer dissociation constant than for aptamer-OTA interactions. The ssDNAs' having less than 18 hydrogen bonds did not affect the aptamer-OTA affinity. The location of ssDNA's complementary site in the aptamer affeced the kinetics of the interaction and retention of OTA-binding in aptamer-ssDNA complexes. The location of the ssDNA site in the aptamer G-quadruplex led to its unfolding. In the presence of OTA, the unfolding process was longer and takes from 20 to 70 min. The refolding in the presence of OTA was possible and depends on the length and location of the ssDNA's complementary site. The location of the ssDNA site in the tail region led to its rapid displacement and wasn't affecting the G-qaudruplex's integrity. It makes the tail region more perspective for the development of ssDNA-based tools using this aptamer.

Multiplex Assay of Viruses Integrating Recombinase Polymerase Amplification, Barcode-Anti-Barcode Pairs, Blocking Anti-Primers, and Lateral Flow Assay.

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.

Recombinase Polymerase Amplification Assay with and without Nuclease-Dependent-Labeled Oligonucleotide Probe.

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.

Rapid Full-Cycle Technique to Control Adulteration of Meat Products: Integration of Accelerated Sample Preparation, Recombinase Polymerase Amplification, and Test-Strip Detection.

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.

Development of lateral flow assay combined with recombinase polymerase amplification for highly sensitive detection of Dickeya solani.

Dickeya solani, one of the most significant bacterial pathogens, infects potato plants, resulting in severe economic damage. In this study, a lateral flow assay (LFA) combined with isothermal DNA amplification was developed for rapid, specific, and sensitive diagnosis of the potato blackleg disease caused by D. solani. Recombinase polymerase amplification (RPA) was chosen for this purpose. Five primer pairs specific to different regions of the D. solani genome were designed and screened. A primer pair providing correct recognition of the target sequence was aligned with the SOL-C region specific to D. solani and flanked by fluorescein (forward primer) and biotin (reverse primer). Lateral flow test strips were constructed to detect DNA amplicons. The RPA-LFA demonstrated a detection limit equal to 14,000 D. solani colony-forming units per gram of potato tuber. This assay provided sensitivity corresponding to the polymerase chain reaction (PCR) but was implemented at a fixed temperature (39 °C) over 30 min. No unspecific reactions with Pectobacterium, Clavibacter, and other Dickeya species were observed. Detection of latent infection of D. solani in the potato tubers by the developed RPA-LFA was verified by PCR. The obtained results confirmed that RPA-LFA has great potential for highly sensitive detection of latent infection.

Urchin peroxidase-mimicking Au@Pt nanoparticles as a label in lateral flow immunoassay: impact of nanoparticle composition on detection limit of Clavibacter michiganensis.

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. Graphical abstract.

Recombinase polymerase amplification combined with a magnetic nanoparticle-based immunoassay for fluorometric determination of troponin T.

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. Graphical abstract Schematic representation of a new kind of immunoassay that is enhanced by making use of recombinase polymerase amplification (immunoRPA). Reporter DNA was conjugated with specific antibodies, then amplified and finally quantified fluorometrically. Limit of detection of troponin T in plasma within 2 h was 9.4 ± 2.1 pg•mL-1.

Enlargement of Gold Nanoparticles for Sensitive Immunochromatographic Diagnostics of Potato Brown Rot.

Lateral flow immunoassay (LFIA) is a convenient tool for rapid field-based control of various bacterial targets. However, for many applications, the detection limits obtained by LFIA are not sufficient. In this paper, we propose enlarging gold nanoparticles' (GNPs) size to develop a sensitive lateral flow immunoassay to detect Ralstonia solanacearum. This bacterium is a quarantine organism that causes potato brown rot. We fabricated lateral flow test strips using gold nanoparticles (17.4 ± 1.0 nm) as a label and their conjugates with antibodies specific to R. solanacearum. We proposed a signal enhancement in the test strips' test zone due to the tetrachloroauric (III) anion reduction on the GNP surface, and the increase in size of the gold nanoparticles on the test strips was approximately up to 100 nm, as confirmed by scanning electron microscopy. Overall, the gold enhancement approach decreased the detection limit of R. solanacearum by 33 times, to as low as 3 × 104 cells∙mL⁻1 in the potato tuber extract. The achieved detection limit allows the diagnosis of latent infection in potato tubers. The developed approach based on gold enhancement does not complicate analyses and requires only 3 min. The developed assay together with the sample preparation and gold enlargement requires 15 min. Thus, the developed approach is promising for the development of lateral flow test strips and their subsequent introduction into diagnostic practice.

Measurement of (Aptamer-Small Target) KD Using the Competition between Fluorescently Labeled and Unlabeled Targets and the Detection of Fluorescence Anisotropy.

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 binding constants of the aptamer-OTA-Flu and the aptamer-OTA complexes were found to be equal to 245 + 33 and 63 + 18 nM, respectively. The value for the aptamer-OTA complexes was confirmed by the equilibrium dialysis technique. The resulting constant was 80 ± 9 nM. The versatility and methodological simplicity of the proposed protocol, as well as the short implementation time, are why it can be recommended as an effective tool for characterizing aptamer-ligand complexes.

The registration of aptamer-ligand (ochratoxin A) interactions based on ligand fluorescence changes.

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.

Post-assay growth of gold nanoparticles as a tool for highly sensitive lateral flow immunoassay. Application to the detection of potato virus X.

This article demonstrates a new kind of a highly sensitive lateral flow immunoassay (LFIA). It is based on the enlargement of the size of gold nanoparticles (GNPs) directly on the test strip after a conventional LFIA. Particle size enlargement is accomplished through the catalytic reduction of HAuCl4 in the presence of H2O2 and through the accumulation of additional gold on the surface of the GNPs. To attain maximal enhancement of the coloration of the zone in the test strip and to achieve a minimal background, the concentration of precursors, the pH value, and the incubation time were optimized. GNPs on the test strip are enlarged from 20 to 350 nm after a 1-min treatment at room temperature. The economically important and widespread phytopathogen potato virus X (PVX) was used as the target analyte. The use of the GNP enlargement method results in a 240-fold reduction in the limit of the detection of PVX, which can be as low as 17 pg·mL-1. The total duration of the assay, including virus extraction from the potato leaves, lateral flow, and the enhancement process, is only 12 min. The diagnostic efficiency of the technique was confirmed by its application to the analysis of potato leave samples. No false positives or false negatives were found. The technique does not depend on specific features of the target analyte, and it is conceivably applicable to numerous GNP-based LFIAs for important analytes. Graphical abstract An enlargement solution (containing HAuCl4 and H2O2) was dripped on the strip after common lateral flow immunoassay. Gold nanoparticles on the strip (20 nm) catalyze gold reduction and the formation of larger particles (up to 350 nm), resulting in a 240-fold lower detection limit within 1 min.

How to Improve Sensitivity of Sandwich Lateral Flow Immunoassay for Corpuscular Antigens on the Example of Potato Virus Y?

A simple approach was proposed to decrease the detection limit of sandwich lateral flow immunoassay (LFIA) by changing the conditions for binding between a polyvalent antigen and a conjugate of gold nanoparticles (GNPs) with antibodies. In this study, the potato virus Y (PVY) was used as the polyvalent antigen, which affects economically important plants in the Solanaceae family. The obtained polyclonal antibodies that are specific to PVY were characterized using a sandwich enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). For LFIA, the antibodies were conjugated with GNPs with a diameter of 17.4 ± 1.0 nm. We conducted LFIAs using GNP conjugates in a dried state on the test strip and after pre-incubation with a sample. Pre-incubating the GNP conjugates and sample for 30 s was found to decrease the detection limit by 60-fold from 330 ng∙mL-1 to 5.4 ng∙mL-1 in comparison with conventional LFIA. The developed method was successfully tested for its ability to detect PVY in infected and uninfected potato leaves. The quantitative results of the proposed LFIA with pre-incubation were confirmed by ELISA, and resulted in a correlation coefficient of 0.891. The proposed approach is rapid, simple, and preserves the main advantages of LFIA as a non-laboratory diagnostic method.

Less is More: A Comparison of Antibody-Gold Nanoparticle Conjugates of Different Ratios.

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.

Development of a lateral flow immunoassay for rapid diagnosis of potato blackleg caused by Dickeya species.

Early detection of potato infections is essential for effective disease management. The aim of this study was to develop a lateral flow immunoassay (LFIA) for rapid detection of a serious potato disease, potato blackleg, caused by Dickeya dianthicola and Dickeya solani. Polyclonal antibodies specific to different strains of Dickeya were obtained from rabbits after immunization with bacterial cells of D. dianthicola and D. solani. Enzyme-linked immunosorbent assay testing with use of a wide range of bacterial species showed that the polyclonal antibodies detect closely related strains of D. dianthicola and D. solani. Cross-reactivity with widespread pathogenic bacteria (nine species) and saprophytes of healthy potato plants was not detected. The LFIA based on the obtained antibodies and gold nanoparticles with average diameter of 20 nm was developed. Under optimized conditions, the LFIA method enabled the analysis of potato extracts within 10 min, with a visual limit of detection of 1 × 105 CFU/ml for leaves and 4 × 105 CFU/ml for tubers. The assay was tested on potato stem and tuber extracts, and the results of the LFIA were confirmed in 92.1% of samples using the real-time polymerase chain reaction. The findings confirmed that the developed LFIA could be used for monitoring blackleg infection without the need for special equipment or skills. Graphical Abstract The developed lateral flow immunoassay is an efficient tool for rapid detection of a serious potato disease, potato blackleg, caused by Dickeya dianthicola and Dickeya solani.

Nonlinear responses to waterborne cadmium exposure in zebrafish. An in vivo study.

Cadmium (Cd) has proved to be associated with numerous toxic effects in aquatic organisms via waterborne exposure. With a view to investigate Cd toxicity along a broad spectrum of exposures reaching from environmental to toxic, we employed adult zebrafish (Danio rerio) for an in vivo study. A number of 10 fish per tank were placed in 40L tanks and were exposed for 30 days to 0.0, 5.0, 25, 50, 75, 100 and 1000µgCd per liter. There were 2 tanks for each Cd exposure (duplicate experiment). Mortality was recorded daily, dead fish were collected and tissue samples were obtained for histologic observation, whereas remaining tissues were stored for Cd burden determination. Surviving fish were collected at the end of the experiment. Median overall survival (OS) in days was found to be 9.0, 11.0, 8.0 and 7.0 for 25µg/L, 50µg/L, 75µg/L and 100µg/L respectively, with all of them showing mortality greater than 50%. Remarkably, fish exposed to the highest Cd concentration (1000µg/L) survived the longest exhibiting a mean OS of 29.2 days. Cd determination in fish tissue was conducted with an in house ICP-MS method and levels ranged from 3.1 to 29.1ng/mg. Log Cd tissue levels were significantly correlated with the log Cd exposure levels (r = 0.535, p < 0.001). The highest Cd burden was determined for fish exposed to 1000µgCd /L (mean = 12.2ng/mg). Histopathology supported these results. Our findings disclose a deviation in toxic responses through the range of Cd concentrations, leading to nonlinear responses. These differentiated responses, could be linked to hormesis phenomena.

Enhancement of lateral flow immunoassay by alkaline phosphatase: a simple and highly sensitive test for potato virus X.

Alkaline phosphatase (ALP) was used as an amplification tool in lateral flow immunoassay (LFIA). Potato virus Ð¥ (PVX) was selected as a target analyte because of its high economic importance. Two conjugates of gold nanoparticles were applied, one with mouse monoclonal antibody against PVX and one with ALP-labeled antibody against mouse IgG. They were immobilized to two fiberglass membranes on the test strip for use in LFIA. After exposure to the sample, a substrate for ALP (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium) was dropped on the test strip. The insoluble dark-violet diformazan produced by ALP precipitated on the membrane and significantly increased the color intensity of the control and test zones. The limit of detection (0.3 ng mL-1) was 27 times lower than that of conventional LFIA for both buffer and potato leaf extracts. The ALP-enhanced LFIA does not require additional preparation procedures or washing steps and may be used by nontrained persons in resource-limited conditions. The new method of enhancement is highly promising and may lead to application for routine LFIA in different areas. Graphical abstract Two gold nanoparticles (GNP) conjugates were used - the first with monoclonal antibodies (mAb) (GNP-mAb); the second - alkaline phosphatase-labeled antibody against mAb (GNP-anti-mAb-ALP). The immuno complexes are captured by the polyclonal antibodies (pAb) in the test zone. Addition of the substrate solution (BCIP/NBT) results in the accumulation of the insoluble colored product and in a significance increase in color intensity.

Unexpected Electrophoretic Behavior of Complexes between Rod-like Virions and Bivalent Antibodies.

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.

Multiarray on a test strip (MATS): rapid multiplex immunodetection of priority potato pathogens.

Multiarray on a test strip (MATS) was developed for the detection of eight important potato pathogens. The proposed assay combines the rapidity of immunochromatography with the high throughput of array techniques. The test zone of the immunochromatographic strip comprises ordered rows of spots containing antibodies specific for different potato pathogens. The assay benefits from the simplicity of immunochromatography; colored immune complexes form at the corresponding spots within the test zone. The presence and intensity of the coloration are used for identification of the target pathogens. The MATS was applied to the simultaneous detection of eight priority potato pathogens, characterized by the following limits of detection: 1 ng/mL for potato virus X and the ordinary type of potato virus Y, 10 ng/mL for potato virus M, 20 ng/mL for potato leaf roll virus, 40 ng/mL for necrotic-type potato virus Y, 100 ng/mL for potato virus S, 300 ng/mL for potato virus A, and 10(4) cells/mL for Clavibacter michiganensis subsp. sepedonicus. Analysis time was 15 min. The observed sensitivity of the MATS was comparable to the traditional enzyme-linked immunosorbent assay. The developed technique was tested on potato leaf extracts, and its efficiency for on-site control of the pathogens was confirmed in 100 % by commercial LFIA test strips. Graphical abstract Location of binding zones in the developed multiarray on a test strip (MATS) for simultaneous detection of eight pathogens.

Neurofilament Light Protein Rod Domain Exhibits Structural Heterogeneity.

Neurofilaments are neuron-specific proteins that belong to the intermediate filament (IFs) protein family, with the neurofilament light chain protein (NFL) being the most abundant. The IFs structure typically includes a central coiled-coil rod domain comprised of coils 1A, 1B, and 2, separated by linker regions. The thermal stability of the IF molecule plays a crucial role in its ability for self-association. In the current study, we investigated the thermal stability of NFL coiled-coil domains by analyzing a set of recombinant domains and their fusions (NFL1B, NFL1A+1B, NFL2, NFL1B+2, and NFLROD) via circular dichroism spectroscopy and differential scanning calorimetry. The thermal stability of coiled-coil domains is evident in a wide range of temperatures, and thermal transition values (Tm) correspond well between isolated coiled-coil domains and full-length NFL. NFL1B has a Tm of 39.4 °C, and its' fusions, NFL1A+1B and NFL1B+2, have a Tm of 41.9 °C and 41.5 °C, respectively. However, in the case of NFL2, thermal denaturation includes at least two thermal transitions at 37.2 °C and 62.7 °C. These data indicate that the continuous α-helical structure of the coil 2 domain has parts with varied thermal stability. Among all the NFL fragments, only NFL2 underwent irreversible heat-induced denaturation. Together, these results unveil the origin of full-length NFL's thermal transitions, and reveal its domains structure and properties.