Analytical Methods DOI: 10.1002/anie.200702690 Indirect Electrochemical Sensing of Radicals and Radical Scavengers in Biological Matrices Fritz Scholz,* Gabriela López de Lara Gonzµlez, Leandro Machado de Carvalho, Maurício Hilgemann, KheniaZ. Brainina, Heike Kahlert, Robert Smail Jack, and DangTruong Minh Free radicals, such as the hydroxyl radical OHC and the superoxide radical O C 2 , belong to the most reactive chemical species known. In organic tissues, a number of enzymes, for example the superoxide dismutases, catalytically destroy these damaging species which are byproducts of the cell metabolism, and known for their potential carcinogenetic action. [1,2] Radicals also play an important role in the organism)s innate immunity. Free oxygen radicals are pro- duced by circulating monocytes and neutrophiles, in response to the lipopolysaccharide (LPS) of the Gram-negative bacterial outer membrane. This radical formation is impor- tant in the destruction of the bacteria after phagocytosis. The activation of these cells must be carefully regulated since excessive radical production may destroy the host)s own tissue and contribute to septic shock. So called antioxidants are compounds known to react with free radicals, inactivating them and thus preventing their cell- damaging action. [3,4] Because of the great importance of free radicals and antioxidants, there is considerable demand for techniques to detect and quantify these two groups of compounds. Electron spin resonance ranks first for radical detection because it is highly selective for the detection of paramagnetic species. [5] In some cases UV/Vis spectroscopy can also be applied to detect certain free radicals. [6] Anti- oxidants on the other hand are usually quantified by their destructive action towards free radicals. Whereas spectro- scopic techniques can be highly selective and sensitive for certain radicals, this is not true in all cases and it is frequently difficult to apply them in situ in chemical or biological systems. A number of highly sensitive electrochemical biosensors for detection of free oxygen radicals and antiox- idants have been reported. [7–11] They are based on the use of immobilized redox proteins, especially cytochrome C, which is easily reduced by the oxygen radicals, and typical catalytic currents can be measured. All these protein-based biosensors suffer from a limited stability, and their preparation is rather time consuming. Herein, we report a completely new approach to detect free radicals using an electrochemical procedure, in which the radicals destroy a well defined molecular layer on an electrode. Self-assembled monolayers (SAMs) of alkylthiols can be easily prepared on the surface of mercury and gold electrodes, and, if suitable compounds are adsorbed, the electrochemical signal of a dissolved redox probe, for example, the hexammine ruthenium(III) complex, can be completely blocked. [12,13] Although these SAMs are known to be stable, we have discovered that they can be rather easily attacked by free radicals. When such an electrode with a SAM is exposed to a solution in which free radicals are generated, for example, by the Fenton reaction [Eq. (1), see for example ref. [14–16]], the free radicals destroy the SAM, and the electrochemical signal of the redox probe recovers to a degree proportional to the extent of dissolution of the SAM. Fe 2þ þ H 2 O 2 ! Fe 3þ þ OH C þ OH  ð1Þ Figure 1 depicts voltammograms recorded at a mercury electrode and at a gold electrode in a solution of [Ru(NH 3 ) 6 ] 3+ before modification of the electrode surface with a SAM of hexanethiol, after SAM formation, and after attacking the SAM with OHC radicals produced in a Fenton solution for 1 minute or for 5 minutes. Figure 2 shows a plot of peak currents versus time of reaction of the SAM with the OHC radicals of the Fenton solution. Control experiments have shown that the SAM is neither attacked by the hydrogen peroxide, nor by iron(II) or iron(III) ions. Experiments with hexacyanoferrate(II) as redox probe and hexanethiol SAM on gold and hexadecanethiol SAMs on gold electrodes revealed that the hexadecanethiol is also attacked by the OHC radicals, although complete removal of the SAM could not be achieved. The removal of SAMs from the electrode can be also achieved by very strong oxidants, such as permanga- nate in acidic solution; however, such reactions cannot [*] Prof. Dr. F. Scholz, G. López de Lara Gonzµlez, Dr. H. Kahlert Institut für Biochemie Universität Greifswald Felix-Hausdorff-Strasse 4, 17487 Greifswald (Germany) Fax: (+ 49)3834-864-451 E-mail: fscholz@uni-greifswald.de Homepage: http://www.chemie.uni-greifswald.de/ ~ analytik/ Prof. Dr. L. Machado de Carvalho, M. Hilgemann Universidade Federal de Santa Maria Departamento de Química Caixa Postal 5051, Santa Maria—RS (Brazil) Prof. Dr. K. Z. Brainina Urals State Economic University Department of Chemistry 8th of March St. 62, Ekaterinburg 620219 (Russia) Prof. Dr. R. S. Jack, D. T. Minh Institut für Immunologie und Transfusionsmedizin Universität Greifswald Sauerbruchstrasse, 17487 Greifswald (Germany) Supporting information for this article is available on the WWW under http://www.angewandte.org or from the author. Angewandte Chemie 8079 Angew. Chem. Int. Ed. 2007, 46, 8079 –8081 # 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim