Contact information

• Office – C05/315
• Phone – 549 49 3816
• Email – gorris@mail.muni.cz

Teaching

• C8116 Immunochemical techniques

UCNPs are lanthanide-doped nanocrystals (e.g. NaYF₄:Yb,Er) that are highly photostable and emit visible light under near-infrared excitation (anti-Stokes emission). Compared to two-photon excitation, UCNPs can be excited under much milder conditions (< 1.000.000-fold) using a continuous 980-nm laser.

Bioanalytical applications require UCNPs that do not aggregate, are highly stable in suspension and have a well-defined surface architecture (Chem. Soc. Rev. 2015). We characterized and purified UCNPs by agarose gel electrophoresis (ACS Appl. Mater. Interfaces 2014, Anal. Chem. 2019) and developed an upconversion scanner for the highly sensitive detection of UCNPs in the gel (Anal. Chem. 2016). Such homogeneous UCNP labels are the foundation for the development of sensitive immunoassays that can be used e.g. for monitoring the pharmaceutical diclofenac with a limit of detection (LOD) of 0.02 ng/mL (Anal. Chem. 2016). We set up an upconversion wide-field microscope with single nanoparticle sensitivity for implementing a single-molecule immunoassay (digital assay, Angew. Chem. Int. Ed. 2020). The digital readout improved the LOD of the cancer marker prostate specific antigen (PSA) by a factor of 16 (23 fg mL^–1, 800 aM) compared to a conventional analog readout (Anal. Chem. 2019). We further developed digital assays for the sensitive detection of cardiac troponin, a marker of myocardial infarction (Adv. Healthc. Mater. 2021), SARS-CoV-2 (Anal. Chem. 2021) and provided a theoretical foundation for the development of digital assays (Anal. Chem. 2022). Another research topic relates to the design of UCNPs as labels in immunohistochemistry (Nanoscale 2020) that have been commercialized by the Swedish company Lumito. We have recently summarized the protocols for the design of UCNPs for cancer detection and imaging (Nature Protocols 2022).

Assoc. Prof. Gorris established and served as the Chair of the COST Action CM1403 “The European Upconversion Network: From the Design of Photon-upconverting Nanomaterials to Biomedical Applications” to support interdisciplinary research on UCNPs. He is also the founder of the first international conference specifically dedicated to the topic of upconversion (UPCON’16 in Wroclaw (highlighted in Nature Photonics 2016) and UPCON’18 in Valencia). The next UPCON’24 is scheduled from 7^th to 11^th 2024 in Montreal.

We have designed large arrays of 250 × 250 (62 500) chambers each defining a volume of 40 femtoliters (fL) in the surface of fused silica slides by photolithography and reactive ion etching. Several hundred individual enzyme molecules are isolated together with an excess of fluorogenic substrate in the chambers of the femtoliter array. According to the Poisson distribution, an enzyme concentration of 1.8 pM results in only one enzyme molecule in every twentieth chamber. Each enzyme molecule turns over a fluorogenic substrate to a fluorescent product, which is recorded in parallel by conventional widefield epifluorescence microscopy. Only chambers that contain a single enzyme molecule light up while empty chambers remain dark.

This detection scheme has enabled us to implement a “three-in-one” enzyme assay (Anal. Bioanal. Chem. 2015): (1) The concentration of the active enzyme is given by the number of fluorescent - i.e. enzyme-occupied - chambers in the array. The ability to determine concentrations by counting individual molecules is called a “digital readout”. (2) In contrast to conventional bulk enzyme assays, the substrate turnover rate of a single enzyme molecule is not influenced by the enzyme concentration. (3) The observation of single enzyme molecules yields information on the catalytic heterogeneity in an enzyme population. The activity of individual enzyme molecules differs strongly among individual molecules in an enzyme population (static heterogeneity), which can be attributed to distinct long-lived conformational states. Matthias Mickert won the Enzyme Assay Award 2016 from the biotech company Novozymes for his role in the development of the single enzyme molecule analysis platform.