Activity of AC electrokinetically immobilized horseradish peroxidase
- 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 stainingDielectrophoresis (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.…
Author details: | Mareike PrüferORCiD, Christian WengerORCiD, Frank Fabian BierORCiDGND, Eva-Maria LauxGND, Ralph HölzelORCiDGND |
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DOI: | https://doi.org/10.1002/elps.202200073 |
ISSN: | 0173-0835 |
ISSN: | 1522-2683 |
Pubmed ID: | https://pubmed.ncbi.nlm.nih.gov/35904497 |
Title of parent work (English): | Electrophoresis : microfluidics, nanoanalysis & proteomics |
Publisher: | Wiley |
Place of publishing: | Hoboken |
Publication type: | Article |
Language: | English |
Date of first publication: | 2022/07/29 |
Publication year: | 2022 |
Release date: | 2024/02/01 |
Tag: | AC electrokinetics; dielectrophoresis; enzyme activity; immobilization;; nanoelectrodes |
Number of pages: | 14 |
First page: | 1920 |
Last Page: | 1933 |
Funding institution: | European Regional Development Fund (ERDF); Brandenburg Ministry of; Science, Research and Cultural Affairs (MWFK) |
Organizational units: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie |
DDC classification: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
Peer review: | Referiert |
Publishing method: | Open Access / Hybrid Open-Access |
License (German): | CC-BY - Namensnennung 4.0 International |