Detection of Hydrogen Peroxide 4/5 – with Prussian Blue

This chapter is part of the series ‘Detection of hydrogen peroxide with Prussian Blue’. This chapter delves deeper into the specifics of detecting hydrogen peroxide using Prussian Blue.

Detecting Hydrogen Peroxide

Electrochemical detection of hydrogen peroxide is interesting for the construction of biosensors using oxidases. The main issue with regards to detection is that an oxidation of hydrogen peroxide takes place at potentials above 0.6 V vs. Ag/AgCl. At potentials that high, many other species often present in real samples might also interfere. These species could be ascorbate (vitamin C), bilirubin, and urate.

Ideas were developed to exclude these species from electrodes by a membrane. This membrane would make all transport processes from and towards the electrode more difficult. Later mediators to transport the electrons from the enzyme to the electrode were developed and hydrogen peroxide detection was obsolete for some oxidase-based electrodes.

As mentioned before (see Chapter Why Detect Hydrogen Peroxide?), hydrogen peroxide is produced by enzymes, but it is hazardous for cells. Cells use peroxidases to deplete hydrogen peroxide. Horseradish peroxidase is popular for electrochemical applications, due to the fact that it is possible for the electrode to directly communicate with the enzyme’s active center. So the peroxidase can be used for hydrogen peroxide detection, but this enzyme’s high costs, low stability, and difficulty to bind it to the electrodes made it interesting to look for inorganic alternatives.

Using Prussian Blue

Thin Prussian Blue layers on electrodes are a very suitable alternative. The first investigations in 1990 showed well-performing sensors by applying around 40 mV vs Ag/AgCl for reduction. The scientific community was highly interested when demonstrations showed that the catalytic activity of Prussian Blue for hydrogen peroxide is 100 times higher than the one for oxygen. Optimization processes lead to Prussian Blue-based sensors that were able to work well at 0 V vs. Ag/AgCl so that Prussian Blue is called “artificial peroxidase”. At these low potentials, many common interfering substances do not show electrochemical reactions.

The catalytic effect and selectivity of Prussian Blue is a consequence of the Prussian Blue’s crystal structure. The crystal has small channels that allow the hydrogen peroxide to enter. Surrounded by the iron ions an electron transfer to the hydrogen peroxide becomes easy. For most of the other interfering species, the channels are not big enough.


Unfortunately, these sensors are limited to neutral or acid solutions. Prussian Blue is not stable in alkaline solutions. The iron ions from the Prussian Blue form iron hydroxide which is soluble in water. This is an issue when the corresponding oxidase has its optimal pH value in the alkaline region. Researchers have developed different ways to create a Prussian Blue film that will show a higher stability in neutral or alkaline media. Most of them are based on the idea that iron is easily accessible for the hydroxide ions, so a change in structure will already lead to a higher stability. Different techniques are new ways of deposition or precipitating Prussian Blue in the presence of other ions.

Sitnikova et al. [1] have developed a layered film of Prussian Blue (iron hexacyanoferrate) and nickel hexacyanoferrate. This film is stable for hours without any loss in sensitivity and it is also stable in neutral as well as alkaline solutions. The presence of other iron ligands also poses no threat to this layer. 


[1] N. Sitnikova, A. Borisova, M. Komkova, A. Karyakin, Anal Chem 83 (2011), p. 2359-2363.