Spectrally and Spatially Multiplexed Serological Array-in-Well Assay Utilizing Two-Color Upconversion Luminescence Imaging

Abstract

We demonstrate a simple dual-mode multiplexed array-in-well immunoassay for simultaneous classification and detection of serum IgG and IgM antibodies against influenza A and human adenoviruses based on the color and position of the upconversion luminescence on the array. Biotinylated influenza A/H1N1 and A/H5N1 as well as adenovirus serotype 2 and 5 hexon antigens along with positive and negative controls were printed in an array format onto the bottom of streptavidin-coated microtiter wells. The anti-influenza A and antiadenovirus antibodies present in the sample were captured to the array and detected with antihuman antibody-coated upconverting nanophosphors (UCNPs). The green emitting UCNPs (NaYF4:Yb(3+),Er(3+)) coated with antihuman IgG and blue emitting UCNPs (NaYF4:Yb(3+),Tm(3+)) coated with antihuman IgM were used to detect human IgG and IgM antibodies, respectively. The emission of the bound UCNPs was imaged free of autofluorescence with anti-Stokes photoluminescence microwell imager. No spectral cross-talk was observed between green and blue emitting UCNPs. Also the cross-reactivities between UCNP-conjugates and immobilized human IgG and IgM antibodies were negligible. Position of the signal on the array defined the antigen specificity and the antibody class was defined by the color of the upconversion luminescence. This technology could be used for differentiation between acute infection from past infection and immunity. Additionally, the class of the antibody response can be used for the differentiation between primary and secondary infections, hence, facilitating epidemiological seroprevalence studies.

Impact of Lanthanide Nanomaterials on Photonic Devices and Smart Applications

“…Moreover, the upconversion colors are tunable through multiple ways, such as simply changing the composition of the nanoparticles and the doped sensitizer/activator couples. Benefiting from the spectral superiority of UCNPs, Soukka and co‐workers developed a dual‐mode multiplex array‐in‐well assay ( Figure ), which facilitates simultaneous detection of immune response against multiple microbial/viral antigens and differentiation of acute infection with IgM positivity from a past IgM negative but IgG positive immune response . Dual‐mode multiplexing by using NaYF 4 :Yb 3+ ,Tm 3+ , and NaYF 4 :Yb 3+ ,Er 3+ UCNPs has also been employed in lateral flow strip (LFS) assays with smartphone detection.…”

“…The images are composite overlays from two separate pseudocolored images. Reproduced with permission . Copyright 2016, American Chemical Society.…”

Advances in Optical Single‐Molecule Detection: En Route to Supersensitive Bioaffinity Assays

“…ther advantages of UCNPs include ah igh photostability and multiple and narrow emission bands that can be tuned individually for the multiplexed detection of several analytes in parallel. [97] Forexample,the group of Soukka [98] developed am ultiplexed array-in-well assay to determine the immune status against virus infections in human serum samples by the spatial arrangement of several virus antigens on am icrotiter plate.A dditionally,t wo types of UCNP labels with different emission colors (Er-doped:g reen;T m-doped:b lue) were used to distinguish between IgG and IgM antibody classes.…”

Surface chemistry modified upconversion nanoparticles as fluorescent sensor array for discrimination of foodborne pathogenic bacteria

“…Recently, the fluorescent sensor arrays constructed from a series of functioned UCNPs probes have exhibited excellent power for identification of analytes (such as phosphate compounds, grape wines, proteins, glycated hemoglobin, vitamin B12, viruses, biotinylated antibodies and human IgG and IgM antibodies) with high classification accuracy in a timely and cost-effective manner [22,[28][29][30][31][32][33]. UCNPs materials have been widely utilized to realize the quantification of single bacteria in previous researches [9][10][11][12]34].…”