Artificial Intelligence-Aided Massively Parallel Spectroscopy of Freely Diffusing Nanoscale Entities

[Image: see text] Massively parallel spectroscopy (MPS) of many single nanoparticles in an aqueous dispersion is reported. As a model system, bioconjugated photon-upconversion nanoparticles (UCNPs) with a near-infrared excitation are prepared. The UCNPs are doped either with Tm(3+) (emission 450 and 802 nm) or Er(3+) (emission 554 and 660 nm). These UCNPs are conjugated to biotinylated bovine serum albumin (Tm(3+)-doped) or streptavidin (Er(3+)-doped). MPS is correlated with an ensemble spectra measurement, and the limit of detection (1.6 fmol L(–1)) and the linearity range (4.8 fmol L(–1) to 40 pmol L(–1)) for bioconjugated UCNPs are estimated. MPS is used for observing the bioaffinity clustering of bioconjugated UCNPs. This observation is correlated with a native electrophoresis and bioaffinity assay on a microtiter plate. A competitive MPS bioaffinity assay for biotin is developed and characterized with a limit of detection of 6.6 nmol L(–1). MPS from complex biological matrices (cell cultivation medium) is performed without increasing background. The compatibility with polydimethylsiloxane microfluidics is proven by recording MPS from a 30 μm deep microfluidic channel.