Cytochrome c biosensor--a model for gas sensing

Abstract

This work is about gas biosensing with a cytochrome c biosensor. Emphasis is put on the analysis of the sensing process and a mathematical model to make predictions about the biosensor response. Reliable predictions about biosensor responses can provide valuable information and facilitate biosensor development, particularly at an early development stage. The sensing process comprises several individual steps, such as phase partition equilibrium, intermediate reactions, mass-transport, and reaction kinetics, which take place in and between the gas and liquid phases. A quantitative description of each step was worked out and finally combined into a mathematical model. The applicability of the model was demonstrated for a particular example of methanethiol gas detection by a cytochrome c biosensor. The model allowed us to predict the optical readout response of the biosensor from tabulated data and data obtained in simple liquid phase experiments. The prediction was experimentally verified with a planar three-electrode electro-optical cytochrome c biosensor in contact with methanethiol gas in a gas tight spectroelectrochemical measurement cell.

Keywords: biosensor; cytochrome c; model; prediction; sensing process; thiol.

Figure 1.

Gas-measurement set-up—A mixture of 100 ppm methanethiol in nitrogen was taken from a pressurized gas bottle. The gas was humidified and could stream into the gas-tight measurement cell. Valves allowed switching between test gas (humidified air with methanethiol) and reference gas (humidified air without methanethiol).The images show the gas-tight spectroelectrochemical measurement cell and a schematic picture of the biosensor.

Figure 2.

Schematic representation of the methanethiol gas biosensor—Methanethiol molecules in the gas phase are the analytes to be detected. The first step in the sensing cascade is the transfer of methanethiol from gas to liquid phase (Step A). Deprotonation of methanethiol occurs after being dissolved in the aqueous liquid phase (Step B). Initially, there is a steep concentration gradient of methanethiol across the liquid phase before methanethiol distributes evenly within the liquid phase (Step C). Cytochrome c that is bound to SnO₂ on FTO reacts with methanethiolate anions, which generates the readout signal (Step D). The symbols in the drawing do not reflect the true scales of the represented parts of the sensor.

Figure 3.

UV-spectra of cytochrome c—typical spectra of cytochrome c with three absorbance peaks of its reduced form (Fe²⁺) at 550 nm, 521 and 414 nm (solid line) and two peaks of its oxidized form (Fe³⁺) at 530 nm and 408 nm (dotted line). The inset shows an expanded view of the spectra between 500 and 580 nm.

Figure 4.

Diffusion gradients—Calculated concentration gradients of methanethiol across the liquid phase at selected time intervals after initial contact of the sample gas with the biosensor.

Figure 5.

Formation rate of cytochrome c (Fe²⁺) in liquid phase plotted vs. mercaptoethanol anion concentrations—Cytochrome c was adsorbed onto SnO₂ on FTO and mercaptoethanol was dissolved in buffer solution. Mercaptoethanolate reduced the oxidized cytochrome c and lead to increased absorption at 550 nm. Changes in concentration of cytochrome c were calculated from changes in light absorption via Lambert-Beer’s law.

Figure 6.

Gas biosensor measurements—with a cytochrome c modified electrode and gaseous methanethiol. The changes in absorbance at 550 nm are plotted versus the time of the experiment. The moments when reference gas was switched to methanethiol-containing gas sample are marked with “ON”, whereas “OFF” indicates switching from methanethiol-containing gas sample to reference gas. The vertical arrow indicates electrochemical oxidation of cytochrome c (60 s, +100 mV vs. Ag/AgCl) to reset to baseline absorbance and to prepare the biosensor for the next exposure of gas sample.