Key significance of DNA-target size in lateral flow assay coupled with recombinase polymerase amplification.

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.

Cadmium, lead and mercury in muscle tissue of gilthead seabream and seabass: Risk evaluation for consumers.

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).

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.

Complex analysis of concentrated antibody-gold nanoparticle conjugates' mixtures using asymmetric flow field-flow fractionation.

Conjugates of gold nanoparticles (GNPs) with antibodies are powerful analytical tools. It is crucial to know the conjugates' state in both the concentrated and mixed solutions used in analytical systems. Herein, we have applied asymmetrical flow field-flow fractionation (AF4) to identify the conjugates' state. The influence of a conjugate's composition and concentration on aggregation was studied in a true analytical solution (a concentrated mixture with stabilizing components). GNPs with an average diameter of 15.3±1.2nm were conjugated by adsorption with eight antibodies of different specificities. We found that, while the GNPs have a zeta potential of -31.6mV, the conjugates have zeta potentials ranging from -5.8 to -11.2mV. Increased concentrations (up to 184nM, OD520=80) of the mixed conjugate (mixture of eight conjugates) did not change the form of fractograms, and the peak areas' dependence on concentration was strongly linear (R2 values of 0.99919 and 0.99845 for absorption signal and light scattering, respectively). Based on the gyration (Rg) and hydrodynamic (Rh) radii measured during fractionation, we found that the nanoparticles were divided into two populations: (1) those with constant radii (Rg=9.9±0.9nm; Rh=14.3±0.5nm); and (2) those with increased radii from 9.9 to 24.4nm for Rg and from 14.3 to 28.1nm for Rh. These results confirm that the aggregate state of the concentrated and mixed conjugates' preparations is the same as that of diluted preparations and that AF4 efficiently characterizes the conjugates' state in a true analytical solution.