Enzyme based biosensors

A large number of proteins and enzymes (cytochrome c, myoglobin, hemoglobin, phosphatase, trypsin,…) have been encapsulated within sol-gel matrices (SiO2/TMOS) since the pioneering work of D. Avnir et al. Even microorganisms such as bacteries (Escherichia coil) or yeast spores (Saccharomyces cerevisiae) have been recently encapsulated within sol-gel silica matrices and preserve their biological activity so that biosensors or biocatalysts could be synthesized via the sol-gel route. The entrapment of biological species is actually more difficult because of denaturation of proteins by alcohol and pH. Encapsulation is often performed at low temperature on ice in order to slow down denaturation reactions

Two steps procedure is usually performed to slow down the denaturation reactions:

•          1- Prehydrolysis of alkoxide precursors:

•           Silicone alkoxides and water are usually mixed in a common solvent like alcohols( ethanol is strictly prohibited whereas small amounts of methanol could be tolerated). TMOS is used as a molecular precursor and prehydrolyzed via acid catalysis by adding an HCl-H2O mixture (pH=2) to the TMOS. Methanol is released upon hydrolysis and behaves as a co-solvent ,a clear fully hydrolyzed solution is readily obtained

2- Encapsulation of proteins:

Proteins would be destroyed at low pH and a buffer has to be added before (or with) the proteins suspension. The pH increase rapidly, up to pH=7, leading to the fast condensation of hydrolyzed precursors. Gelation then occurs within a few minutes and proteins remain trapped with the growing oxide network.

Glucose oxidase (GOD) has been often chosen as a model for such studies. This enzyme is stable and highly reactive for catalyzing the oxidation of D-glucose into β-D- gluconolactone while oxygen is reduced into hydrogen peroxide as fallows:

C6H12O6 + O2 GOD C6H10O6 + H2O2
                              glutconolactone

This reaction is now used for the detection of glucose and the commercial production of glucose biosensors.

Glucose can be detected via either optical or electrochemical measurements.

The enzymatic oxidation of glucose can also be detected via electrochemical means. Direct electron transfers between electrode and the active site of GOD are not possible because of the steric hindrance of the large GOD biomolecule

A ferrocence mediator (FC) has to be used as fallows:

            GODred  + FCox               GODox + FCred

•          FCox accepts electrons from GODred to form FCred which diffuses to the electrode where it is reoxidized. Detection is performed via cyclic voltammetry measurements. The electrochemical current is given by the oxidation of ferrocence and the magnitude of the current is proportional to glucose concentration.

Enzyme based electrochemical sensors have been made by P.Audebert et al via the immobilization of GOD in sol-gel silica matrices.