High amplification rates from the association of two enzymes confined within a nanometric layer immobilized on an electrode: modeling and illustrating example.

Electrochemical responses (e.g., chronoamperometric) obtained with an immobilized enzyme that produces an electroactive species may be used to quantitate the amount of enzyme or the concentration of its substrate. It is shown, on theoretical and experimental bases, that product-to-substrate coupling with a second enzyme co-immobilized with the first within one or within a small number of monolayers, allows high amplification rates (higher than 1000), avoids membrane transport limitations, and lends itself to precise kinetic analyses that provide guidelines for optimization of the analytical sensitivity. Very large amplification factors, as large as several thousands, can be reached experimentally, in agreement with appropriately derived theoretical predictions, thus opening the route to the rational design of high-performance substrate sensing or affinity assays applications.