Researchers Develop a Biosensor for Measuring Extracellular Hydrogen Peroxide Concentrations

Several processes in the human body are regulated by biochemical reactions involving hydrogen peroxide (H₂O₂). Although it can act as a 'secondary messenger', relaying or amplifying certain signals between cells, H₂O₂ is generally toxic because of its oxidant character.

The latter means that it converts (oxidizes) biochemical molecules like proteins and DNA. The oxidizing property of H₂O₂ is of potential therapeutic relevance for cancer, though: deliberately causing tumor cells to increase their H₂O₂ concentration would be a way to destroy them.

In light of this, but also for monitoring pathologies associated with H₂O₂ overproduction, it is crucial to have a means to reliably quantify hydrogen peroxide concentrations in the extracellular environment. Now, Leonardo Puppulin from Nano Life Science Institute (WPI-NanoLSI), Kanazawa University and colleagues have developed a sensor for measuring concentrations of H₂O₂ in the vicinity of cell membranes, with nanometer-resolution.

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.