Photon-upconversion nanoparticles as background-free luminescent labels in single molecule immunoassays

• Applicant: Professor Dr. Joachim Wegener, since 9/2021
• Subject Area: Analytical Chemistry
• Term: from 2015 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 277934007

Single molecule analysis has attracted broad interest because it promises to reach the ultimate detection limit of analytical chemistry. In recent years, great progress has been made in fluorescence-based single molecule detection. Analytical applications, however, are impeded by the high background signal of fluorescence detection. The aim of this project is to establish a single molecule immunoassay, which circumvents the current limitations of single molecule techniques and is much more sensitive compared to a conventional immunoassays: (1) The high background fluorescence can be avoided by using photon-upconversion nanoparticles (UCNPs) as a detection label. UCNPs can be excited under near-infrared illumination, which strongly reduces autofluorescence and light scattering of surrounding molecules. (2) A much larger sample volume can be probed by using a pre-concentration step on the surface of microspheres. Such microspheres can be loaded into femtoliter arrays and read out one-by-one. During the first part of research project, we set up an upconversion microscope for detecting UCNPs at the single nanoparticle level and developed a single molecule upconversion-linked immunoassay (ULISA) for the cancer marker prostate specific antigen (PSA). The single molecule ULISA has a limit of detection (LOD) of 1.2 pg mL−1 PSA in 25 % blood serum, which is about ten times more sensitive compared to commercial immunoassays (published in Analytical Chemistry 2017, 89, 11825-11830). We also prepared large arrays consisting of tens of thousands of femtoliter wells to load individual microspheres into the arrays and register them one-by-one. In the second part of the research project, we will switch from the microtiter plate immunoassay to the microsphere-based assay in order to further improve the sensitivity of the single molecule ULISA. Microspheres covered by an analyte-specific antibody will be suspended in an analyte sample to immobilize the analyte efficiently on the microsphere surface. After binding of the UCNP detection system, the microspheres will be loaded onto femtoliter arrays and registered individually. It is important to interrogate large numbers of microspheres because only few microspheres carry an analyte molecule if the analyte concentration in a sample is low. The femtoliter array will allow for detecting the presence and the absence of a single immune complex on the surface of each microsphere, thus indicating single analyte molecules. We will adapt the microsphere assay for the multiplexed detection of several analytes in parallel and the detection of single virus particles in wastewater samples.

• DFG Programme: Research Grants
• Ehemaliger Antragsteller: Professor Dr. Hans-Heiner Gorris, until 8/2021