Highly sensitive multiplex lateral flow immunoassay of phytopathogens using Au@Pt nanoparticles as the colorimetric and catalytic label

A device for multiplex lateral flow immunoassay (LFIA) of major viral (potato viruses X and Y, potato leafroll virus) and bacterial (Ralstonia solanacearum, Clavibacter michiganensis, Dickeya solani, Pectobacterium atrosepticum) potato pathogens was developed. The device includes test strips connected with the central fiberglass reservoir. The addition of the probe (300 µL) to the reservoir initiates the migration of the liquid along membranes and the formation of immune complexes labeled with Au@Pt nanosized label. Besides the own Au@Pt coloration, the colorimetric signals are enhanced using the peroxidase-mimicking activity of Au@Pt. The developed device facilitated rapid (15 min), multiplex (7 targets), and highly sensitive LFIA for viral (330-480 pg/mL) and bacterial (500-104 cells/mL) pathogens. © 2021 Author(s)

DEVELOPMENT OF LATERAL FLOW IMMUNOASSAY WITH DOUBLE ENHANCEMENT FOR PLANT VIRUS DETECTION: A CASE STUDY WITH POTATO VIRUS X

Crop loss due to pathogens is one of the main reason of food shortage. Potato virus X is one of the main phytopathogens of potatoes, losses from it can reach up to 40%. Thus, a rapid and sensitive method for its early detection is necessary and modern methods of disease diagnosis can significantly reduce potato yield losses. The aim of this study is to develop lateral flow immunoassay (LFIA) with amplification for the detection of PVX in low concentrations. Two types of amplification were used: 1) magnetic concentration of magnetic nanoparticles (MNPs) conjugated with antibodies specific to PVX, 2) MNP aggregates based on gold nanoparticles (GNPs). The MNPs conjugates with biotinylated antibodies and GNPs conjugates with streptavidin were synthesized to realize these approaches. The LFIA with double enhancement (LOD 0.25 ng/mL) was found out to be 32-fold more sensitive compared to LFIA without enhancement (LOD 8 ng/mL). The developed LFIA with double enhancement was effective for the detection PVX in leaf extracts and for the quantitative analysis results from 0.25 to 125 ng/mL (based on the densitometry of line colouration)

Double-enhanced lateral flow immunoassay for potato virus X based on a combination of magnetic and gold nanoparticles

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. © 2017 Elsevier B.V

Double-enhanced lateral flow immunoassay for potato virus X based on a combination of magnetic and gold nanoparticles

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. © 2017 Elsevier B.V

Comparative study of four coloured nanoparticle labels in lateral flow immunoassay

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. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

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

Comparative study of magnetic beads and microplates as supports in heterogeneous amplified assay of miRNA-141 by using mismatched catalytic hairpin assembly reaction

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 × 109 particles/mL, with a surface area of 65 mm2. The identical surface area was used upon the assay performance with polystyrene microplates. Comparison of MBs- and microplate-assays for miRNA-141 determination showed that the obtained calibration curves and their detection limit values were the same and did not depend on the used carrier. The results showed that the choice of a carrier for heterogeneous assays should be guided by the convenience of the assay performance, not its surface area. © 2022 Elsevier B.V

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. © 2019 by the authors. Licensee MDPI, Basel, Switzerland

New lateral flow immunoassay for on-site detection of Erwinia amylovora and its application on various organs of infected plants

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. © 2021 Elsevier Lt