The FluoRox principle: A redox protein or enzyme is immobilized on an electrode through a linker and labeled with a fluorescent label (FL). Excitation light hits the label and the absorbed energy is emitted in the form of fluorescence. The fluorescence intensity is quenched or not, depending on the redox state of the active centre (++). A change in redox state of the active centre leads to a change in fluorescence. The redox state changes when the active centre exchanges electrons (e–) either with the electrode or with the analyte.
The operating principle of typical electrochemical biosensors (e.g., the glucose sensor) is based on direct current detection. A leap in sensitivity can be achieved by fluorescence detection, which has reached the point that the observation of single molecules under suitable conditions is feasible. The EdRox Network aims to exploit a new concept that can lead to such a super-sensitive biosensor, which we call FluoRox. This concept not only has the promise of increasing sensitivity by orders of magnitude, but it also allows miniaturization to the sub-micrometer level, thereby opening up new areas of technological applications.
In the FluoRox principle (see Figure 1; European patent application PCT 04258120.7; further applications pending) the redox protein is labeled with a fluorescent dye. We have found that in many cases the fluorescence intensity depends strongly on the oxidation state of the redox protein or enzyme (Schmauder et.al., J. Biol. Inorg. Chem. 10 (2005) 683; Kuznetsova et al., Anal. Biochem. (2006) in press). The label lights up or dims if a redox event takes place: the fluorescence signals the passage of electrons on their way from the electrode to the active site of the enzyme or vice versa. Therefore, we can monitor enzyme activity by following fluorescence; instead of electrons we monitor photons, i.e., detection is transferred from the electrochemical to the optical domain. We can therefore utilize the superior sensitivity and efficiency of optical detectors.
Four practical steps have to be taken to realize a working FluoRox device:
- Engineering FluoRox proteins/enzymes. Many redox proteins and enzymes can be used as selective and sensing elements in a FluoRox sensor but they must be tailored for fluorescent labeling and surface immobilization.
- Engineering FluoRox electrodes. Electrodes on which the proteins/enzymes are to be immobilized should be transparent as well as conducting, and be accessible for optical techniques. Such electrodes are not commercially available, and must be constructed.
- Functional characterization of FluoRox electrodes. Once the protein/enzyme has been successfully immobilized on an electrode, the surface has to be characterized and the activity of the protein/enzyme has to be tested.
- Development of the FluoRox sensor. After a suitable electrode has been modified with a functional, immobilized protein layer, fluorescent detection of redox events has to be established, eventually at the single molecule level. A specialized optical set-up has to be constructed for this purpose.
The research efforts will concentrate a) on the production of proteins/enzymes and their adaptation for FluoRox purposes and b) on the construction of electrodes with specially prepared surfaces. The immobilization of proteins on the electrodes will be pursued, and the constructs will be characterized in terms of integrity and functionality by, e.g., electrochemical analysis, fluorescence imaging and spectroscopy. The topographical characterization of functionalized electrodes involves advanced scanning probe techniques, such as atomic force microscopy (AFM), scanning tunneling microscopy (STM), and electrochemical imaging. Further exploration deals with single molecule detection of redox events on the electrodes, and on the completion of a prototype of a FluoRox sensor. The efforts will concentrate on a demonstration of proof-of-principle and an assessment of the sensitivity of the Fluorox sensor.