Quantifying the Numbers of Gold Nanoparticles in the Test Zone of Lateral Flow Immunoassay Strips

Abstract

Lateral flow immunoassay (LFIA) is a rapid and simple point-of-care method for the detection of various analytes. In the colorimetric sandwich format, the reaction outcome is a colored test zone line formed by Au nanoparticle (AuNP) conjugates captured by bound analyte molecules. Although nanoparticle design is crucial for the assay sensitivity, the correlation between the test zone brightness and the number and size of captured AuNPs has not been studied in detail. To fill this gap, we used an unprecedented set of 10 spherical and monodisperse AuNPs with diameters d ranging from 16 to 115 nm. The calculated optical properties are in excellent agreement with two-layered Mie theory for Au cores coated with 3 nm CTAC shells with a refractive index of 1.5. Different concentrations of AuNPs (ICP-MS and UV–vis measurements) were spotted onto a nitrocellulose LFIA membrane, and the color intensity of the spots was measured and analyzed with RGB and HSV color parameters. The minimal detected spot intensity was proportional to the surface nanoparticle density and the particle volume. The derived size dependence means that extinction rather than scattering is the main physical mechanism behind spot brightness. The limit of detection (LOD) in terms of the surface nanoparticle density scaled like the inverse third power of the particle size (more precisely, like ∼d –3.1) and was about 7 × 107 and 1.5 × 105 particles/mm2 for 16 and 115 nm AuNPs, respectively. We analyzed an ideal LFIA format, when one analyte molecule delivers just one AuNP to the test zone. In this case, the theoretical LODs were in the pg/mL range for a typical LFIA format with 0.1 mL of a 25 kDa analyte. In practice, these estimations could be increased by 2 orders of magnitude because of the larger ratio of analyte to captured AuNPs. This strongly reduced the assay sensitivity to the the ng/mL level. Although an ideal LFIA predicts a strong increase in the assay sensitivity with the AuNP size (scales like ∼d 3.1), this improvement could be compensated for in part by an increase in the number of ineffective analyte molecules bound to the AuNP surface (scales like ∼d 2).

Advantages of Highly Spherical Gold Nanoparticles as Labels for Lateral Flow Immunoassay

“…The presented data differ with the earlier published [37] comparison of GNPs with Au cores and small CTAC shells that cover the diameter range from 16 to 115 nm with high homogeneity (RSD 2−3%). In the previous study, the monotonous dependence of LoD on the GNP diameter was found to reach 0.157 pg/mL of cTnI for the largest GNPs.…”

“…Some experimental results show that 30-40 nm GNPs are optimal [17,35,36] because smaller particles have unacceptably small extinction cross-sections and small surface areas for antibody internalization. On the other hand, we recently demonstrated that increasing the nanoparticle size up to 115 nm reduced the LoD [37]. Moreover, conclusions about the capabilities of a label can be made only by comparing the analytical characteristics achieved for its conjugates with antibodies of different compositions [38][39][40][41][42].…”

Surface-Enhanced Raman Scattering-Based Lateral-Flow Immunoassay

“…To meet both requirements, one needs a detection sensitivity of about 0.01 ng/L, which is higher than the reported SERS-based data. Recent analysis [ 11 ] showed that, in an ideal LFIA format, one analyte molecule delivers just one Au NP to the test zone. In this case, the theoretical LODs were in picograms per milliliter range for a typical LFIA format with 0.1 mL of a 25 kDa analyte.…”

“…We recently estimated the required particle densities, depending on their size. So, for particles with a size of 16 nm, it is necessary that the biospecific reaction delivers at least 6.7 × 10 7 particles/mm 2 to the test zone [ 11 ]. This substantial surface density explains one of the first drawbacks of LFIA—low sensitivity relative to other immunological methods.…”

Gold Nanoparticle-Coated Microspheres for Enhancing Immunosensor Detection of Hepatitis B Virus Surface Antibody

“…The number of gold nanoparticles per analyte capturing area might be the key of this enhanced detection. For AuNP*, the limit of detection was approximated to the particle diameter to the power of negative three (d -3 ), meaning the bigger AuNP* would result in less detection limit [12]. However, one analyte was captured by the limited number of AuNP*, so there was not much gold nanoparticles per area.…”