A biosensor for measuring extracellular hydrogen peroxide concentrations

The biosensor consists of a gold nanoparticle with organic molecules attached to it. The whole cluster is designed so that it anchors easily to the outside of a cell's membrane, which is exactly where the hydrogen peroxide molecules to be detected are. As attachment molecules, the scientists used a compound called 4MPBE, known to have a strong Raman scattering response: when irradiated by a laser, the molecules consume some of the laser light's energy. By measuring the frequency change of the laser light, and plotting the signal strength as a function of this change, a unique spectrum is obtained -- a signature of the 4MPBE molecules. When a 4MPBE molecule reacts with a H₂O₂ molecule, its Raman spectrum changes. Based on this principle, by comparing Raman spectra, Puppulin and colleagues were able to obtain an estimate of the H₂O₂ concentration near the biosensor.

After developing a calibration procedure for their nanosensor -- relating the H₂O₂ concentration to a change in Raman spectrum in a quantitative way is not straightforward -- the scientists were able to produce a concentration map with a resolution of about 700 nm for lung cancer cell samples. Finally, they also succeeded in extending their technique to obtain measurements of the H₂O₂ concentration variation across cell membranes.

Puppulin and colleagues conclude that their "novel approach may be useful for the study of actual H₂O₂ concentrations involved in cell proliferation or death, which are fundamental to fully elucidate physiological processes and to design new therapeutic strategies."

Surface-enhance Raman spectroscopy

The biosensor developed by Leonardo Puppulin from Kanazawa University and colleagues is based on a method called surface-enhanced Raman spectroscopy (SERS). The principle derives from Raman spectroscopy, in which differences between the incoming and the outcoming frequencies of laser light irradiated onto a sample are analyzed. The spectrum obtained by plotting the signal strength as a function of frequency difference is characteristic for the sample, which can in principle be a single molecule. Typically, however, the signal coming from one molecule is too weak to detect, but the effect can be enhanced when the molecule is absorbed on a rough metal surface. Puppulin and colleagues applied the technique to (indirectly) detect hydrogen peroxide; their Raman-responsive molecule is a compound called 4MPBE, which is modified when exposed to hydrogen peroxide.