Enhanced lateral flow immunoassay using gold nanoparticles loaded with enzymes

Abstract

The use of gold nanoparticles (AuNPs) as labeling carriers in combination with the enzymatic activity of the Horseradish Peroxidase (HRP) in order to achieve an improved optical lateral flow immunoassay (LFIA) performance is here presented.Briefly in a LFIA with an immune-sandwich format AuNPs are functionalized with a detection antibody already modified with HRP, obtaining an "enhanced" label. Two different detection strategies have been tested: the first one following just the red color of the AuNPs and the second one using a substrate for the HRP (3 different substrates are evaluated), which produces a darker color that enhances the intensity of the previous red color of the unmodified AuNPs. In such very simple way it is gained sensitivity (up 3

Enhancing the Signal of Lateral Flow Immunoassays by Using Different Developing Methods

“…Previous strategies to improve lateral flow immunoassay performance have included adopting a two-step assay for enzymatic colorimetric signal generation or prestabilization of reactants. (2,15,16) Our results showed a dot intensity increase similar to that obtained by Parolo et al, (2) who demonstrated a 3-or 4-fold intensity increase by adopting a two-step assay using an enzymatic colorimetric signal, although their system also increased the analytical sensitivity by an order of magnitude. Changing the strip architecture resulted in a 2-or 3-fold change in signal intensity.…”

“…Results indicated the signal intensity was improved for both the anti-Cy5 and anti-Texas Red tests at DNA concentrations greater than 0.01 μM; this improvement was significant for the anti-Cy5 test at 10 and 1 μM DNA (>100% increase; p < 0.05), and for the anti-Texas Red test at 1 μM DNA (42% increase, p < 0.05). However, while increased the loading of GNP conjugate is common, (2,3) it dramatically increased the cost per strip, since GNP conjugates are the most expensive reagent in the assay. We were thus interested in determining if other running methods could mimic the fully absorbed method while reducing the amount of waste associated with simply providing an excess of unbound GNP conjugates.…”

“…(1) However, achieving low analytical sensitivity with a visual colorimetric signal is one of the limitations of these devices. For sandwich lateral flow immunoassays that use a hapten and antibody detection system, the analytical sensitivity is reported to be improved through the use of enzyme substrates, (2) dual-gold nanoparticle conjugates, (3) thermal contrast, (4) and changes to the device architecture. (5) While each of these methods improved the signal intensity and analytical sensitivity, they also increased the complexity of the detection system.…”

Multicolor immunochromatographic strip test based on gold nanoparticles for the determination of aflatoxin B1 and fumonisins

“…This effort is testified also by the number of papers reporting new materials to be used as labels [10][11][12][39][40], sometimes in combination with innovative detection technologies [13,41], and of enhancement strategies aimed at increasing the sensitivity of the ICST compared to using traditional gold nanoparticles [42][43], as also summarized in two comprehensive reviews [44][45]. Within the number of novel materials for labeling antibodies in ICTS, the request for analytical devices with multiplexing capability is prompting towards using labels easily distinguishable from each other, such as nanoparticles with different colors [19][20]23].…”

Recent Advances in Nanoparticles-based Lateral Flow Biosensors

“…Conversely, in the absence (or lower than the cutoff value) of target analyte, numerous colored reagent-labeled analyte would bind on test zone, and two characteristic red lines can be observed from the test zone and control zone respectively. On the other hand, when the target analyte owns more than one specific antibodies or DNA aptamers, sandwich format could be employed to test the target analyte [64][65][66][67][68][69][70]33,71]. Figure 2 presents the typical DNA aptamers-based sandwich format.…”