Single Molecule Upconversion-Linked Immunosorbent Assay with Extended Dynamic Range for the Sensitive Detection of Diagnostic Biomarkers

Abstract

The ability to detect disease markers at the single molecule level promises the ultimate sensitivity in clinical diagnosis. Fluorescence-based single-molecule analysis, however, is limited by matrix interference and can only probe a very small detection volume, which is typically not suitable for real world analytical applications. We have developed a microtiter plate immunoassay for counting single molecules of the cancer marker prostate specific antigen (PSA) using photon-upconversion nanoparticles (UCNPs) as labels that can be detected without background fluorescence. Individual sandwich immunocomplexes consisting of (1) an anti-PSA antibody immobilized to the surface of a microtiter well, (2) PSA, and (3) an anti-PSA antibody-UCNP conjugate were counted under a wide-field epifluorescence microscope equipped with a 980 nm laser excitation source. The single-molecule (digital) upconversion-linked immunosorbent assay (ULISA) reaches a limit of detection of 1.2 pg mL (42 fM) PSA in 25% blood serum, which is about ten times more sensitive than commercial ELISAs, and covers a dynamic range of three orders of magnitude. This upconversion detection mode has the potential to pave the way for a new generation of digital immunoassays.

How Nanoparticles Transform Single Molecule Measurements into Quantitative Sensors

“…As such, this becomes a really important question as in many of the systems discussed above, the single molecule binding pair appears to have an unlimited lifetime. This is certainly the case for not only the SiMoA system11a and the nanopore blockade sensor where the nanoparticles are confined into small volumes but also for systems described above where nanoparticles simply bind to a surface 10a,19,28,29b,c. In all cases, where long lifetimes are observed however, the nanoparticles have many receptors conjugated.…”

“…A detection range between 0.02 × 10 −12 and 2 × 10 −12 m was achieved. Exploiting recent developments in nanoparticles with upconversion optical properties, Farka et al have led to a single molecule immunosorbent assay for digital analysis of prostate‐specific antigen (PSA) (Figure b‐i). The strength of upconverting nanoparticles is they can serve as background‐free reporters due to their narrow emission spectra and low‐autofluorescence background, thus rising to the challenge of obtaining sufficient signal above noise.…”

Emerging Technologies and Platforms for the Immunodetection of Multiple Biochemical Markers in Osteoarthritis Research and Therapy

“…Furthermore, the combination of nanotags and characteristic spectrum of the target molecule makes SERS detection a very attractive strategy to achieve multiplexing as recently demonstrated (94,95). Photonupconversion nanoparticles have been used to develop a microtiter plate immunoassay capable of detecting PSA at 1.2 pg/ml, which is 10 times more sensitive than commercial ELISA and covers a dynamic range of three orders of magnitude (96). Such technology has the potential to develop into a new generation of digital immunoassays.…”

<p>Up-Conversion Luminescent Nanoparticles for Molecular Imaging, Cancer Diagnosis and Treatment</p>

“…There were different biomarkers reported; including carcinoembryonic antigen (CEA), 48 carbohydrate antigen (eg, CA19-9), 49 alpha fetoprotein (AFP), 50 and tumor-associated antigens (eg, prostate-specific membrane antigen, PSMA). 51,52 Tumor-associated antigens are specifically located on cell membrane or in intracellular structure; including epidermal growth factor receptor (EGFR), 53 folate receptor-α (FR-α), 54,55 vascular endothelial growth factor receptor (VEGFR), 56 estrogen receptor (ER), and progesterone receptor (PR). These biomarkers are potential for targeted therapy and imaging of cancer.…”