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.

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.

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.

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.

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.