TY  - JOUR
A1  - Prüfer, Mareike
A1  - Wenger, Christian
A1  - Bier, Frank Fabian
A1  - Laux, Eva-Maria
A1  - Hölzel, Ralph
T1  - Activity of AC electrokinetically immobilized horseradish peroxidase
JF  - Electrophoresis : microfluidics, nanoanalysis & proteomics
N2  - Dielectrophoresis (DEP) is an AC electrokinetic effect mainly used to manipulate cells. Smaller particles, like virions, antibodies, enzymes, and even dye molecules can be immobilized by DEP as well. In principle, it was shown that enzymes are active after immobilization by DEP, but no quantification of the retained activity was reported so far. In this study, the activity of the enzyme horseradish peroxidase (HRP) is quantified after immobilization by DEP. For this, HRP is immobilized on regular arrays of titanium nitride ring electrodes of 500 nm diameter and 20 nm widths. The activity of HRP on the electrode chip is measured with a limit of detection of 60 fg HRP by observing the enzymatic turnover of Amplex Red and H2O2 to fluorescent resorufin by fluorescence microscopy. The initial activity of the permanently immobilized HRP equals up to 45% of the activity that can be expected for an ideal monolayer of HRP molecules on all electrodes of the array. Localization of the immobilizate on the electrodes is accomplished by staining with the fluorescent product of the enzyme reaction. The high residual activity of enzymes after AC field induced immobilization shows the method's suitability for biosensing and research applications.
KW  - AC electrokinetics
KW  - dielectrophoresis
KW  - enzyme activity
KW  - immobilization;
KW  - nanoelectrodes
Y1  - 2022
U6  - https://doi.org/10.1002/elps.202200073
SN  - 0173-0835
SN  - 1522-2683
SP  - 1920
EP  - 1933
PB  - Wiley
CY  - Hoboken
ER  - 
TY  - JOUR
A1  - Kruse, Marlen
A1  - Altattan, Basma
A1  - Laux, Eva-Maria
A1  - Grasse, Nico
A1  - Heinig, Lars
A1  - Möser, Christin
A1  - Smith, David M.
A1  - Hölzel, Ralph
T1  - Characterization of binding interactions of SARS-CoV-2 spike protein and DNA-peptide nanostructures
JF  - Scientific reports
N2  - Binding interactions of the spike proteins of the severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) to a peptide fragment derived from the human angiotensin converting enzyme 2 (hACE2) receptor are investigated. 
The peptide is employed as capture moiety in enzyme linked immunosorbent assays (ELISA) and quantitative binding interaction measurements that are based on fluorescence proximity sensing (switchSENSE). 
In both techniques, the peptide is presented on an oligovalent DNA nanostructure, in order to assess the impact of mono- versus trivalent binding modes. 
As the analyte, the spike protein and several of its subunits are tested as well as inactivated SARS-CoV-2 and pseudo viruses. While binding of the peptide to the full-length spike protein can be observed, the subunits RBD and S1 do not exhibit binding in the employed concentrations. 
Variations of the amino acid sequence of the recombinant full-length spike proteins furthermore influence binding behavior. The peptide was coupled to DNA nanostructures that form a geometric complement to the trimeric structure of the spike protein binding sites. 
An increase in binding strength for trimeric peptide presentation compared to single peptide presentation could be generally observed in ELISA and was quantified in switchSENSE measurements. Binding to inactivated wild type viruses could be shown as well as qualitatively different binding behavior of the Alpha and Beta variants compared to the wild type virus strain in pseudo virus models.
Y1  - 2022
U6  - https://doi.org/10.1038/s41598-022-16914-9
SN  - 2045-2322
VL  - 12
IS  - 1
PB  - Macmillan Publishers Limited, part of Springer Nature
CY  - London
ER  -