Thin-Film Transistor-Based Biosensors for Determining Stoichiometry of Biochemical Reactions

Abstract

The enzyme kinetic in a biochemical reaction is critical to scientific research and drug discovery but can hardly be determined experimentally from enzyme assays. In this work, a charge-current transducer (a transistor) is proposed to evaluate the status of biochemical reaction by monitoring the electrical charge changes. Using the malate-aspartate shuttle as an example, a thin-film transistor (TFT)-based biosensor with an extended gold pad is demonstrated to detect the biochemical reaction between NADH and NAD+. The drain current change indicates the status of chemical equilibrium and stoichiometry.

Fig 1. Structure of the TFT-based biosensor and the experimental flow.

(A) Structure and cross-section of the TFT-based biosensor. (B) 8-MOA, EDC/NHS and target protein and ligand were applied to sensing pad step by step. (C) From (b), I_DS-V_GS transfer curves of a bare TFT device along with the current response after the step-by-step application of 8MOA, EDC/NHS and soln. (4). (D) From (c), normalized drain currents at V_GS = 8V and V_DS = 5V can be derived. The drain current of after dispensing EDC/NHS is normalized to 1. The average current increment of soln. (4) is about 17.7% compared to that of EDC/NHS.

Fig 2. Percentage drain current changes at different experimental stages and illustration of the sensing mechanism.

(A) Comparison of current changes of PBS, soln. (1), (2), (3) and (4). (B) Illustration of the incomplete reaction when only NADH and OAA are presented in the solution. Since no (or very small amount of) NAD+ is produced, the TFT device doesn’t sense significant amount of current changes. (C) For a nearly complete reaction when MDH catalyzes NADH and OAA to NAD⁺ and malate. NAD⁺ molecules carrying positive charges are captured by cross linker layer, contributing to the drain current increase. Negative charges are induced in the IGZO channel layer through the Au sensing pad separated by the passivation SiO₂ from the channel.

Fig 3. Experimental results: Comparisons of the corresponding drain current changes; and optical spectrometry measurement of various solutions.

(A) For the nearly complete reaction, the drain current decreases when the concentration is reduced. (B) In comparison, for the incomplete reaction (soln. (7) and (8)), the tenfold increase or decrease of NADH concentration results in a small amount of drain current change. (C) Absorption spectra of pure NADH; along with solutions of nearly complete reaction for comparisons. (D) Absorption spectra of solutions of incomplete reaction are plotted for comparisons.