Synergistic Use of Gold Nanoparticles (AuNPs) and “Capillary Enzyme-Linked Immunosorbent Assay (ELISA)” for High Sensitivity and Fast Assays

Abstract

Using gold nanoparticles (AuNPs) on “capillary enzyme-linked immunosorbent assay (ELISA)”, we produced highly sensitive and rapid assays, which are the major attributes for point-of-care applications. First, in order to understand the size effect of AuNPs, AuNPs of varying diameters (5 nm, 10 nm, 15 nm, 20 nm, 30 nm, and 50 nm) conjugated with Horseradish Peroxidase (HRP)-labeled anti-C reactive protein (antiCRP) (AuNP•antiCRP-HRP) were used for well-plate ELISA. AuNP of 10 nm produced the largest optical density, enabling detection of 0.1 ng/mL of CRP with only 30 s of incubation, in contrast to 10 ng/mL for the ELISA run in the absence of AuNP. Then, AuNP of 10 nm conjugated with antiCRP-HRP (AuNP•antiCRP-HRP) was used for “capillary ELISA” to detect as low as 0.1 ng/mL of CRP. Also, kinetic study on both 96-well plates and in a capillary tube using antiCRP-HRP or AuNP•antiCRP-HRP showed a synergistic effect between AuNP and the capillary system, in which the fastest assay was observed from the “AuNP capillary ELISA”, with its maximum absorbance reaching 2.5 min, while the slowest was the typical well-plate ELISA with its maximum absorbance reaching in 13.5 min.

Functionalized Gold Nanoparticles for the Detection of C-Reactive Protein

“…However, POC assays are still limited in terms of sensitivity and quantitation, when compared to laboratory-based diagnosis, which constrains their application. Commercially available LFA assays (Cholestech (Hayward, CA, USA), Milenia Biotec (Gießen, Germany), BÜHLMANN (Schönenbuch, Switzerland)) are unable to detect CRP concentrations below 0.2 mg·L −1 [ 83 ]. Efforts have been performed to enhance the performance of LFA tests in CRP detection, by improving the configuration of ELISA based assays (e.g., “capillary ELISA” [ 54 ], three-line LFA [ 13 ] and 3 test-line assay [ 55 ]) incorporating tailored size functionalized Au NPs [ 84 ] or using a combined approach [ 83 ].…”

“…Commercially available LFA assays (Cholestech (Hayward, CA, USA), Milenia Biotec (Gießen, Germany), BÜHLMANN (Schönenbuch, Switzerland)) are unable to detect CRP concentrations below 0.2 mg·L −1 [ 83 ]. Efforts have been performed to enhance the performance of LFA tests in CRP detection, by improving the configuration of ELISA based assays (e.g., “capillary ELISA” [ 54 ], three-line LFA [ 13 ] and 3 test-line assay [ 55 ]) incorporating tailored size functionalized Au NPs [ 84 ] or using a combined approach [ 83 ]. “Capillary ELISA” assays performed with Au NPs 10 nm in size conjugated with Horseradish Peroxidase (HRP)-labelled anti-C reactive protein (anti-CRP) were faster and more sensitive than conventional ELISA assay, allowing to detect 0.1 ng·mL −1 CRP after 30 s incubation ( Figure 11 a).…”

“…“Capillary ELISA” assays performed with Au NPs 10 nm in size conjugated with Horseradish Peroxidase (HRP)-labelled anti-C reactive protein (anti-CRP) were faster and more sensitive than conventional ELISA assay, allowing to detect 0.1 ng·mL −1 CRP after 30 s incubation ( Figure 11 a). By contrast, conventional ELISA barely recognized 10 ng·mL −1 of CRP ( Figure 11 b) [ 83 ]. This excellent performance was ascribed to a synergistic effect between Au NPs and the capillary system.…”

Biosensor Based Immunoassay: A New Approach for Serotyping of Toxoplasma gondii

“…They can become the next generation of diagnostic tools, as they show great sensitivity and specificity, replacing with advantage conventional molecular and serological methods [ 16 ]. AuNPs have been used for signal enhancement in order to improve the sensitivity and assay time of some classical methods, such as enzyme-linked immunosorbent assays (ELISA) [ 17 ]. In spite of the progress made thus far using nanotechnology to develop novel, simple and rapid diagnostic tests, only a few methods based on AuNPs have been reported for the detection of parasites [ 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ].…”

Development of a gold-nanorod-based lateral flow immunoassay for a fast and dual-modal detection of C-reactive protein in clinical plasma samples

“…Currently, routine CRP checks in clinical analyses are mainly performed by chemiluminescence, immunoturbidimetry and enzyme-linked immunoassay (ELISA). [9][10][11][12][13] Unfortunately, all of these methods require expensive instruments, which greatly limit their popularizing in resource-poor regions; in addition, their operation processes are complicated, time-consuming and need highly trained personnel, which make them not suitable for postoperative monitoring and point-of-care testing (POCT). 14 Therefore, it is necessary to establish an analytical technique for fast, simple, specic, and accurate quantication and user-friendly detection of CRP in clinical diagnosis.…”