Gorris group

Developing sensitive bioanalytical techniques with the ultimate goal to detect single molecules of an analyte. Focused on research using photon-upconversion nanoparticles and single-molecule analyses in femtoliter volumes.

Dr. rer. nat. Hans-Heiner Gorris

Group leader

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MUNI | ResearchGate

About Hans-Heiner Gorris

Contact information

  • Office – C05/211
  • Phone – 549 49 6035
  • Email – gorris@mail.muni.cz

Curriculum vitae

  • Since 2021 Assistant Professor, Department of Biochemistry, Faculty of Science, Masaryk University
  • Since 2016 Heisenberg fellow of the DFG
  • Since 2015 Habilitation / Private lecturer (PD)
  • Since 2009 Assistant Professor, Institute of Analytical Chemistry, Chemo- and Biosensors at the University of Regensburg
  • 20062009 Post-doctoral fellow with Prof. David R. Walt, Department of Chemistry at Tufts University, USA
  • 2005 PhD, University of Lübeck
  • 20012005 Graduate student with PD Dr. Andreas Frey, Institute of Infectiology in Münster and the Division of Mucosal Immunology at the Research Center Borstel
  • 2001 Diploma, Institute of Infectiology, ZMBE, Münster
  • 19942001 Studies in Biology, University of Münster and University of York, England

Teaching

  • C8116 Immunochemical techniques

Research area

Finding a single molecule of an analyte in a complex biological matrix.

The Gorris group develops sensitive bioanalytical techniques with the ultimate goal to detect single molecules of an analyte in a much more robust and reproducible way than it is currently possible. It is almost impossible to find a single fluorescent molecule among billions of autofluorescent and light scattering molecules in the same detection volume by standard wide-field fluorescence microscopy. Even worse, real biological samples such as blood are notorious for strong (optical) interference. We have explored two ways out of this classic “needle in a haystack” problem: We use the high catalytic power of enzymes for fluorescence signal amplification – a scheme adopted from the well-known enzyme-linked immunosorbent assay (ELISA). Thousands of fluorescent product molecules enclosed in a femtoliter-sized chamber highlight the presence of a single enzyme molecule. The alternative way leads us to the “optical window”, a spectral region between ~650 and 1000 nm, where optical background interference from biological matrices is largely absent. The unique photophysical properties of photon-upconversion nanoparticles (UCNPs) provide access to the optical window because they can be excited under near-infrared (NIR, 980 nm) light and emit light of shorter wavelengths (anti-Stokes emission).

Main research topics

Analyzing single enzyme molecules in femtoliter arrays

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.

(Top left) The SEM image shows a section of the large femtoliter array (250 × 250 wells) and a homogeneous cylindrical shape of each well (4 µm diameter, 3 µm depth). (Top right) Single molecule substrate turnover. Individual molecules of an enzyme population (E1 - En) are isolated in the femtoliter array. Individual enzyme molecules hydrolyze a non-fluorescent substrate to fluorescent resorufin (orange). The substrate turnover of hundreds of individual enzyme molecules is recorded in separate femtoliter chambers by wide-field fluorescence microscopy. Individual substrate turnover rates are then assembled as histograms to expose the activity distribution within the enzyme population (JACS 2014). (Bottom) Digital readout of the enzyme concentration. Solutions of (A) 0.36 pM, (B) 0.9 pM, (C) 1.8 pM, and (D) 3.6 pM β-galactosidase and 100 µM of a fluorogenic substrate are enclosed in a femtoliter arrays. Each chamber occupied by a single enzyme molecule can be distinguished from empty chambers. (E) At low concentrations, the percentage of chambers containing a single enzyme molecule is linearly dependent on the enzyme concentration (Anal. Bioanal. Chem. 2015).

Femotliter arrays have enabled us to address fundamental questions of enzyme kinetics at the single molecule level: (1) Single molecule studies of horseradish peroxidase revealed a particular two-step reaction mechanism involving a radical intermediate that was previously not observed in bulk experiments (JACS 2009, Analyst 2011). (2) Single molecule inhibition studies have yielded new insights into enzyme-inhibitor interactions. If a competitive inhibitor in a concentration close the inhibition constant (Ki) is added to an ensemble reaction, the enzyme activity is reduced to 50 %. In contrast, inhibitor binding or unbinding from a single enzyme molecule are observable as stochastic events (PNAS 2009, Chem. Sci. 2014). (3) In vitro evolution by random mutation and selection can yield new catalytic activities of enzymes. We have compared the kinetics of wildtype and in vitro-evolved β-glucuronidase (GUS) at the single molecule level to gain new insights into molecular evolution. The broader activity distribution of in vitro-evolved GUS compared to the wildtype indicates a higher conformational flexibility of evolved enzymes, which enables the evolved enzyme to catalyze a broader range of different substrates (JACS 2014). (4) We developed a new kinetic framework based on transition state theory to account for protein dynamic effects and heterogeneities in enzyme catalysis (J. Phys. Chem. B 2018).

Photon-upconversion nanoparticles (UCNPs)

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. We have used the multiple emission bands of UCNPs to develop new optical encoding strategies for detecting many analytes in parallel (multiplexing) (Adv. Mater. 2011, Angew. Chem. Int. Ed. 2013).

Bioanalytical applications require UCNPs that do not aggregate, are highly stable in suspension and have a well-defined surface architecture (Chem. Soc. Rev. 2015). Therefore, we characterized and purified UCNPs by agarose gel electrophoresis (ACS Appl. Mater. Interfaces 2014) 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, Microchim. Acta 2017). We set up an upconversion wide-field microscope with single nanoparticle sensitivity for implementing a single molecule immunoassay (digital assay). The digital readout improved the LOD of the cancer marker prostate specific antigen (PSA) by a factor of ten (1.2 pg/mL) compared to a conventional analog readout (Anal. Chem. 2017). We collaborate with the Czech Academy of Sciences in Brno and the University of Turku on these projects.

(Left) TEM images of (A) oleic acid-capped UCNPs and (B) UCNPs with carboxylated silica shell. (C) Agarose gel electrophoresis of (I) carboxylated UCNPs and (II) UCNP-antibody conjugates in duplicates. The arrow indicates the starting point of electrophoresis.

(Right) Single molecule sandwich immunoassay for the detection of PSA. Individual PSA molecules can be counted (digital readout) using UCNPs as a luminescent label (green spots). The calibration curve shows a detection limit of 1.2 pg/mL (Anal. Chem. 2017).

 

Dr. Gorris has established and serves 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, 10, 567-569) and UPCON’18 in Valencia).

Gorris group in 2021

Group members

PhD students: Julian Brandmeier, ​
Master’s students: 
Bachelor students: 

PhD graduates: Andreas Sedlmeier, Raphaela Liebherr

 

Recent publications

An overview of all publications can be found on the website MUNI.

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