Application Note 166: Probing Redox Interactions Between Microbial EPS and p-Nitrophenol Using Electrochemical Surface Plasmon Resonance

Microbial extracellular polymeric substances (EPS) with redox functional groups play a vital role in the bioconversion of pollutants, which can affect their reactivity toward diverse environmental pollutants. However, the redox interactions between different EPS and pollutants have not been addressed till date due to the absence of essential analytical methodologies. In this study, researchers from the University of Science and Technology, China used BI-4500 SPR with the EC-DualFlow^TM analysis module to investigate the interactions between EPS and an aromatic pollutant named p-nitrophenol (PNP) by simultaneously monitoring the electrochemical reaction and their binding kinetics.¹

Figure 1: The schematic diagram of the developed EC-SPR system.

Surface plasmon resonance (SPR) is a real-time, label-free, and concise method for probing biomolecular binding events and their kinetics.²  By combining electrochemical (EC) technology with SPR, the electrochemical interface process can be thoroughly explored.³ When an electrochemical reaction occurs, the thickness of the gold film changes which triggers variations in the SPR resonance angle (refractive index), and is recorded by the detector (Figure 1).⁴ In this study, the  EC SPR system served as a powerful tool to investigate the interactions between EPS at various redox states and an organic pollutant PNP by simultaneously monitoring the electrochemical reaction and the binding kinetics.

Figure 2: a) SPR sensorgrams  and  b) Nyquist plots of the interaction between EPS and PNP.

Table 1 presents the binding parameters (k_a, k_d , and KA) for various redox states of  EPS. The binding affinity of EPS to PNP is related to the redox states of EPS, with this order of EPS_red > EPS_raw > EPS_ox.

Binding  Parameters	EPSred	EPSraw	EPSox
ka (L/g/s)	8.741 × 10− 3	6.573 × 10− 4	4.836 × 10− 4
kd (1/s)	2.753 × 10− 4	2.674 × 10− 4	4.465 × 10− 4
KA (L/g)	31.75	2.458	1.083
Rct (ohm)	1.319 × 105	1.978 × 105	2.547 × 105

EPS reduced PNP to p-aminophenol by donating electrons, and the reductive process depended on the redox states of EPS, determined by their electron donating capacity. The binding rate of EPS_red was significantly higher than that of EPS_raw and EPS_ox, while the dissociation rate of the three forms was similar (Figure 2a). The association signal of EPS_red with PNP increased to reach equilibrium rapidly, resulting in k_a and KA values of 8.741 × 10^{− 3} Liters per gram per second (L/g/s) and 31.75 Liters per gram (L/g), which were ten times higher than those of EPS_raw and EPS_ox. The equilibrium affinity constant (KA) for the binding reaction mentioned in this paper is calculated by dividing k_a by k_d. In the Nyquist plot in (Figure 2b) of the impedance spectrum, the charge transfer resistance (R_ct) value was associated with the diameter of the semicircle observed at higher frequencies. EPS_red showed the lowest charge transfer resistance, indicating that the redox re-action is more readily occurring between them. Both EPS_red and EPS_raw transfer electrons to the nitro group in PNP, resulting in the trans-formation of the nitro group to amino group. On the contrary, EPS_ox exhibited the highest R_ct value indicating the lack of electron transfer to PNP. By employing electrochemical SPR experiments, this study clarified the difference in the reduction of PNP by EPS at different redox states and showed that the reduced state of EPS has the maximum ability to transfer electrons to PNP. This work contributes to a comprehensive understanding of the critical role of EPS redox property in the conversion of refractory pollutants like PNP in biological wastewater treatment systems.

Author: Nguyen Ly and Miyuki Thirumurthy | Biosensing Instrument | Published June 18th, 2025