Upconversion Cross‐Correlation Spectroscopy of a Sandwich Immunoassay

Abstract

Fluorescence correlation and cross‐correlation spectroscopy (FCS/FCCS) have enabled biologists to study processes of transport, binding, and enzymatic reactions in living cells. However, applying FCS and FCCS to samples such as whole blood and plasma is complicated as the fluorescence bursts of diffusing labels can be swamped by strong autofluorescence. Here we present cross‐correlation spectroscopy based on two upconversion nanoparticles emitting at different wavelengths on the anti‐Stokes side of a single excitation laser. This upconversion cross‐correlation spectroscopy (UCCS) approach allows us to completely remove all Stokes shifted autofluorescence background in biological material such as plasma. As a proof of concept, we evaluate the applicability of UCCS to a homogeneous sandwich immunoassay for thyroid stimulating hormone measured in buffer solution and in plasma.

Disentangling optically activated delayed fluorescence and upconversion fluorescence in DNA stabilized silver nanoclusters

“…in single molecule studies) or complicate signal quantification. Methods developed to mitigate some of these problems are two-photon microscopy,1 lanthanide-based upconversion fluorescence,2,3 time-gated imaging,4,5 and optical modulation techniques,6–8 among others. Recently, Fleischer et al introduced optically activated delayed fluorescence (OADF), which can successfully remove unwanted background fluorescence 9,10.…”

Probing heterogeneity of NIR induced secondary fluorescence from DNA-stabilized silver nanoclusters at the single molecule level

“…Such approaches are based on fluorescence upconversion, fluorescence modulation, spectral discrimination and time-gating. [3][4][5][6][7][8][9][10] Recently, Fleicher et al introduced the concept of optically activated delayed fluorescence (OADF, see Fig. 1A), which combines some of the advantages of upconversion and time-gating.…”

Intrinsic anti-Stokes emission in living HeLa cells

“…The anti-Stokes emission signals overlap well with conventional auto-fluorescence from nonstained HeLa cells. Our results also demonstrate that attention must be paid to ensure that intrinsic anti-Stokes emission is accounted for, especially when using other anti-Stokes imaging modalities like upconversion emission from lanthanide based nanoparticles [40], opticallyactivated delayed fluorescence [33] or coherent two-photon absorption microscopy [24,41].…”