Evaluation of a biohybrid photoelectrochemical cell employing the purple bacterial reaction centre as a biosensor for herbicides

Abstract

The Rhodobacter sphaeroides reaction centre is a relatively robust and tractable membrane protein that has potential for exploitation in technological applications, including biohybrid devices for photovoltaics and biosensing. This report assessed the usefulness of the photocurrent generated by this reaction centre adhered to a small working electrode as the basis for a biosensor for classes of herbicides used extensively for the control of weeds in major agricultural crops. Photocurrent generation was inhibited in a concentration-dependent manner by the triazides atrazine and terbutryn, but not by nitrile or phenylurea herbicides. Measurements of the effects of these herbicides on the kinetics of charge recombination in photo-oxidised reaction centres in solution showed the same selectivity of response. Titrations of reaction centre photocurrents yielded half maximal inhibitory concentrations of 208nM and 2.1µM for terbutryn and atrazine, respectively, with limits of detection estimated at around 8nM and 50nM, respectively. Photocurrent attenuation provided a direct measure of herbicide concentration, with no need for model-dependent kinetic analysis of the signal used for detection or the use of prohibitively complex instrumentation, and prospects for the use of protein engineering to develop the sensitivity and selectivity of herbicide binding by the Rba. sphaeroides reaction centre are discussed.

Keywords: Atrazine; Biosensor; Herbicide; Photocurrent; Photovoltaics; Reaction centre.

Fig. 1

Overlay of the D, E and de ɑ-helices that form the Q_B pocket, and terbutryn occupying the pocket in the Rba. sphaeroides reaction centre (yellow (online) or light-grey (print)) and the T. elongatus PSII (cyan (online) or dark-grey (print)). Prepared using Protein Data Bank entries 2BNP (Katona et al., 2005) and 3PRQ (Broser et al., 2010), and PyMOL (Schrödinger, LLC). For a stereo, colour representation see Supplementary Fig. 1.

Fig. 2

Effects of relatively high concentrations of herbicides on reaction centre function. (a) Impact on photocurrent density. To adjust for small variations in absolute current output from cell to cell, traces are normalised to the maximum photocurrent generated by each cell over an identical time period before the addition of the herbicide or inhibitor. Grey background indicates periods without illumination. (b) Effect on charge recombination in photo-excited reaction centres, measured through recovery of primary donor absorbance at 865 nm after photobleaching with a pulse of white light. Raw data (faded lines) are overlaid with fitted single or double exponential decays (solid lines). Data for bentazon, bromoxynil and capsaicin, which were similar to those for the uninhibited and DCMU samples, have been omitted for clarity. Parameters from fits are shown in Supplementary Table 1.

Fig. 3

Photocurrent attenuation by increasing concentrations of terbutryn. Grey background indicates periods without illumination.

Fig. 4

Inhibition of photocurrent generation by atrazine, terbutryn and stigmatellin. Percentage inhibition is the maximum photocurrent density during 30 s illumination relative to that obtained prior to addition of the first aliquot of inhibitor. Each data point is an average from three titrations on three separate cells, with error bars showing the standard error. Solid lines show fits to a logistic function to determine the IC₅₀ (see text).