A label-free electrochemical immunosensor based on decorated cellulose nanofibrous membrane for point-of-care diagnosis of amanitin poisoning via human urine

Abstract

α-Amanitin (AMN) is one of the deadliest toxins from mushrooms, present in the deadly mushroom species Amanita phalloides. It is a bicyclic octapeptide and represents up to 40% of the amatoxins in mushrooms, damaging the liver and kidneys. Current methods of detecting amatoxins are time-consuming and require the use of expensive equipment. A novel label-free electrochemical immunosensor was successfully developed for rapid detection of α-amanitin, which was fabricated by immobilization of anti-α-amanitin antibodies onto a functionalized cellulose nanofibrous membrane-modified carbon screen-printed electrode. An oxidation peak of the captured amanitin on the tethered antibodies was observed at 0.45 V. The performance of the nanofibrous membrane on the electrode and necessary fabrication steps were investigated by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Due to its unique structural features and properties such as high specific surface area and microporous structure, the nanofibrous membrane as an immunosensor matrix for antibody tethering improved the electrochemical performance of the immunosensor by more than 3 times compared with cast membranes. Under the optimal conditions, the assembled immunosensor exhibited high sensitivity toward α-amanitin detection in the range of 0.009-2 ng mL^-1 with a limit of detection of 8.3 pg mL^-1. The results clearly indicate that the fabricated nanofiber-based-immunosensor is suitable for point-of-care detection of lethal α-amanitin in human urine without any pretreatment within 30 min.

Fig. 1

(A) Scheme of the deacetylation process of cellulose acetate. (B) FT-IR spectra of (a) Cel-A NFM and (b) regenerated Cel NFM. (C) Scheme of grafting of citric acid onto Cel NFM. (D) FT-IR spectra of (a) Cel NFM and (b) Cel NFM decorated with citric acid.

Fig. 2

(a) SEM image and (b) diameter distribution of cellulose acetate NFM. (c) SEM image and (d) diameter distribution of Cel NFM.

Fig. 3

(A) CV scans of 2.5 mM [Fe(CN)6]3−/4− at a scan rate of 25 mV s−1 for (a) Cel CM/SPE and (b) Cel NFM/SPE. (B) EIS Nyquist plots in 2.5 mM [Fe(CN)6]4−/3− for (a) bare SPE, (b) Cel NFM (0.05 mm)/SPE, (c) Cel NFM (0.1 mm)/SPE, and (d) Cel NFM (0.2 mm)/SPE. (C) CV scan of AMN (1 μg mL−1) on SPE.

Fig. 4

Response to 1 ng mL−1 AMN of immunosensors fabricated by using different experimental conditions: (a) antibody concentration, (b) antibody immobilization time, (c) immunoreaction temperature, (d) immunoreaction time, and (e) pH of electrolyte solution.

Fig. 5

(a) Electrocatalytic current responses of the fabricated electrochemical immunosensor for the detection of different concentrations of AMN in the range of 9 pg mL−1 to 2 ng mL−1; (b) calibration curve of the immunosensor for the detection of different concentrations of AMN (n = 3).

Scheme 1

Fabrication process and sensing mechanism of the electrochemical immunosensor for AMN detection.