Effect of Particle Size and Surface Chemistry of Photon Upconversion Nanoparticles on Analog and Digital Immunoassays for Cardiac Troponin

Sensitive immunoassays are required for troponin, a low-abundance cardiac biomarker in blood. In contrast to conventional (analog) assays that measure the integrated signal of thousands of molecules, digital assays are based on counting individual biomarker molecules. Photon-upconversion nanoparticles (UCNP) are an excellent nanomaterial for labeling and detecting single biomarker molecules because their unique anti-Stokes emission avoids optical interference, and single nanoparticles can be reliably distinguished from the background signal. Here, the effect of the surface architecture and size of UCNP labels on the performance of upconversion-linked immunosorbent assays (ULISA) is critically assessed. The size, brightness, and surface architecture of UCNP labels are more important for measuring low troponin concentrations in human plasma than changing from an analog to a digital detection mode. Both detection modes result approximately in the same assay sensitivity, reaching a limit of detection (LOD) of 10 pg mL−1 in plasma, which is in the range of troponin concentrations found in the blood of healthy individuals

Effect of Particle Size and Surface Chemistry of Photon-Upconversion Nanoparticles on Analog and Digital Immunoassays for Cardiac Troponin

Sensitive immunoassays are required for troponin, a low-abundance cardiac biomarker in blood. In contrast to conventional (analog) assays that measure the integrated signal of thousands of molecules, digital assays are based on counting individual biomarker molecules. Photon-upconversion nanoparticles (UCNP) are an excellent nanomaterial for labeling and detecting single biomarker molecules because their unique anti-Stokes emission avoids optical interference, and single nanoparticles can be reliably distinguished from the background signal. Here, the effect of the surface architecture and size of UCNP labels on the performance of upconversion-linked immunosorbent assays (ULISA) is critically assessed. The size, brightness, and surface architecture of UCNP labels are more important for measuring low troponin concentrations in human plasma than changing from an analog to a digital detection mode. Both detection modes result approximately in the same assay sensitivity, reaching a limit of detection (LOD) of 10 pg mL(-1) in plasma, which is in the range of troponin concentrations found in the blood of healthy individuals

Influence of Label and Solid Support on the Performance of Heterogeneous Immunoassays

Conventional immunochemical methods used in clinical analysis are often not sensitive enough for early-stage diagnosis, resulting in the need for novel assay formats. Here, we provide a detailed comparison of the effect of different labels and solid supports on the performance of heterogeneous immunoassays. When comparing three types of streptavidin-modified labelshorseradish peroxidase, carboxyfluorescein, and photon-upconversion nanoparticles (UCNPs)UCNPs led to the most sensitive and robust detection of the cancer biomarker prostate-specific antigen. Additionally, we compared the immunoassay formats based on conventional microtiter plates and magnetic microbeads (MBs). In both cases, the highest signal-to-background ratios and the lowest limits of detection (LODs) were obtained by using the UCNP labels. The MB-based upconversion-linked immunosorbent assay carried out with a preconcentration step provided the lowest LOD of 0.46 pg/mL in serum. The results demonstrate that the use of UCNPs and MBs can significantly improve the sensitivity and working range of heterogeneous immunoassays for biomarker detection

Competitive upconversion-linked immunoassay using peptide mimetics for the detection of the mycotoxin zearalenone

Due to increasing food safety standards, the analysis of mycotoxins has become essential in the food industry. In this work, we have developed a competitive upconversion-linked immunosorbent assay (ULISA) for the analysis of zearalenone (ZEA), one of the most frequently encountered mycotoxins in food worldwide. Instead of a toxin conjugate conventionally used in competitive immunoassays, we designed a ZEA mimicking peptide extended by a biotin-linker and confirmed its excellent suitability to mimic ZEA by nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) analysis. Upconversion nanoparticles (UCNP, type NaYF4:Yb,Tm) served as background-free optical label for the detection of the peptide mimetic in the competitive ULISA. Streptavidinconjugated UCNPs were prepared by click reaction using an alkyne-PEG-neridronate linker. The UCNP conjugate clearly outperformed conventional labels such as enzymes or fluorescent dyes. With a limit of detection of 20 pg mL-1 (63 pM), the competitive ULISA is well applicable to the detection of ZEA at the levels set by the European legislation. Moreover, the ULISA is specific for ZEA and its metabolites (alpha and beta-zearalenol) without significant cross-reactivity with other related mycotoxins. We detected ZEA in spiked and naturally contaminated maize samples using liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) as a reference method to demonstrate food analysis in real samples