An integrated relay/nitrate reductase field-effect transistor for the sensing of nitrate (NO₃-)

An integrated enzyme-functionalized field-effect transistor (ENFET) device for the sensing of nitrate ions is described. An aminosiloxane-functionalized gate interface is modified with N-methyl-N′-(carboxyalkyl)-4,4′-bipyridinium relay units. The complex formed between nitrate reductase and the bipyridinium units on the gate surface is crosslinked with glutaric dialdehyde to yield a stable relay-enzyme layer on the gate interface. In the presence of sodium dithionite as electron donor, the biocatalyzed reduction of nitrate to nitrite ion is stimulated. The ratio between the oxidized and reduced states of the bipyridinium sites regulates the gate potential, and is controlled by the concentration of NO₃- ions in the system. The effect of the chain length tethering the N-methyl-N′-(carboxyalkyl)-4,4′-bipyridinium units to the gate surface on the biocatalyzed reduction of NO₃- ions, and on the NO₃- FET sensor performance is discussed. The devices that include the bipyridinium units tethered to the gate interface with methylene chain length, -(CH₂)_n, where n ≥ 7, reveal a detection limit of 7 × 10^-5 M for nitrate and a sensitivity of 52 ± 2 mV dec^-1. The response time of the device is as low as 50 s, and the operational time of the system is ca. 85 s. We estimate the surface coverage of nitrate reductase on the gate surface to be ca. 1.2 × 10^-12 mol cm^-2.