Analysis of the Biochemical Reaction Status by Real-Time Monitoring Molecular Diffusion Behaviors Using a Transistor Biosensor Integrated with a Microfluidic Channel

Abstract

Traditional methods of monitoring biochemical reactions measure certain detectable reagents or products while assuming that the undetectable species follow the stoichiometry of the reactions. Here, based upon the metal-oxide thin-film transistor (TFT) biosensor, we develop a real-time molecular diffusion model to benchmark the concentration of the reagents and products. Using the nicotinamide adenine dinucleotide (NADH)-oxaloacetic acid with the enzyme of malate dehydrogenase as an example, mixtures of different reagent concentrations were characterized to extract the ratio of remaining concentrations between NAD⁺ and NADH. We can thus obtain the apparent equilibrium constant of the reaction, (8.06 ± 0.61) × 10⁴. Because the whole analysis was conducted using a TFT sensor fabricated using a semiconductor process, our approach has the advantages of exploring biochemical reaction kinetics in a massively parallel manner.

Figure 1

Transient drain current profile of a bare TFT with the microfluidic channel filled with PBS (black line). The current response profile of the TFT when the 0.01 × PBS solution is injected to the microfluidic channel at t = 200 s (red line) is shown. The corresponding turbulence occurs at around 250–350 s.

Figure 2

(a) Transient drain current responses of NADH solutions of different concentrations. (b) Transient drain currents of NAD⁺ solutions of different concentrations. Note that those marked in red are the POI. (c) Curve fitting of NADH in the POI. (d) Curve fitting of NAD⁺ in the POI.

Figure 3

(a) Correlation of B and the concentration of NADH; (b) Correlation of B and the concentration of NAD⁺. The equation of the correlation is shown in the figure.

Figure 4

Transient drain current response when injecting 10 mM of OAA into the microfluidic channel.

Figure 5

(a) Transient drain current responses of various mixtures and (b) corresponding fitting curves in the POI.

Figure 6

Biosensor employed in this work for monitoring biochemical reaction. The sensor is composed of a TFT in the left and a microfluidic channel in the right. ROI is located on the Au sensing pad, which is connected to the top gate through Au bond wires.

Figure 7

Illustration of the diffusion of target molecules in the microfluidic channel. The electrical charges carried by the molecules above the Au sensing pad will be detected by the TFT.