Flexible and Printed Electrochemical Immunosensor Coated with Oxygen Plasma Treated SWCNTs for Histamine Detection.

Heterocyclic amine histamine is a well-known foodborne toxicant (mostly linked to "scombroid poisoning") synthesized from the microbial decarboxylation of amino acid histidine. In this work, we report the fabrication of a flexible screen-printed immunosensor based on a silver electrode coated with single-walled carbon nanotubes (SWCNTs) for the detection of histamine directly in fish samples. Biosensors were realized by first spray depositing SWCNTs on the working electrodes and by subsequently treating them with oxygen plasma to reduce the unwanted effects related to their hydrophobicity. Next, anti-histamine antibodies were directly immobilized on the treated SWCNTs. Histamine was detected using the typical reaction of histamine and histamine-labeled with horseradish peroxidase (HRP) competing to bind with anti-histamine antibodies. The developed immunosensor shows a wide linear detection range from 0.005 to 50 ng/mL for histamine samples, with a coefficient of determination as high as 98.05%. Average recoveries in fish samples were observed from 96.00% to 104.7%. The biosensor also shows good selectivity (less than 3% relative response for cadaverine, putrescine, and tyramine), reproducibility, mechanical and time stability, being a promising analytical tool for the analysis of histamine, as well as of other food hazards.


Immunosensor Development
The immunosensor realization was based on a direct enzyme-linked immunosorbent assays (ELISA) principle, where the antihistamine antibodies were placed onto the oxygen plasma OP treated single-walled carbon nanotubes (SWCNTs)-modified working electrode (WE). For the competitive reaction a mixture of 4 µL of the free His (diluted on PBS or fish extract) and 4 µL Hishorseradish peroxidase (HRP) was placed onto the WE ( Figure S1). The competitive reaction between the free His and the His-HRP to bind with the anti-histamine antibody was performed for 2 h at 37 °C . Figure S1. Scheme of His immunosensor developed on flexible screen-printed sensor.
In particular, the expected reaction (separately also qualitatively confirmed by an expected color change), is: H2O2 + HRP(red) → HRP(ox) + H2O HRP(ox) + TMB(red) → HRP(red) + TMB(ox) TMB(ox) + 2H + → TMB(red) The electrode thickness was measured by a non-contact 3D-optical profilometer (ProFilm3D from Filmetrics, Unterhaching, Germany). The 2D profile for the thickness measurement is given in Figure SX, where the thickness was measured in terms of step height. The step height of the silver electrode was 5.38 µm.

Optimization of Spray Deposited SWCNTs Layers
To optimize the number of spray deposited layers on oxygen plasma (OP) treated silver electrode, cyclic voltammetry (CV) was performed. In this experiment, 50, 100, 150 and 200 SWCNTs layers were spray deposited on the OP-treated electrode, which were subsequently covered with 50 µl of 1 mM [Fe(CN)6] 3-/4-containing 0.1 M KCl. Finally, CV measurements were performed. It was found that 100 layers of spray deposited SWCNTs layers show the highest oxidation/reduction current, 45.61% enhancement on oxidation current and 76.12% enhancement on reduction current as compared to OP treated bare electrode. The oxidation/reduction current and the enhancement of the current generation as compared to the OP treated bare electrode are presented in Table S1.

Fourier-Transform Infrared Spectroscopy Characterization of SWCNTs
Besides the higher current generation, the OP treatment leads to the formation of carbonyl and/or carboxylic groups as reported in the literature [1]. The presence of these groups can improve the immobilization of antibodies on the surface of SWCNTs. To confirm the presence of these polar groups we have analyzed the SWCNTs before and after OP treatment using a FT-IR INVENIO-R e ATR spectrophotometer (Bruker), operating in transmittance mode to record the spectra over 400-4000 cm −1 . Figure S2 shows Fourier-transform infrared (FTIR) spectra of a) spray deposited SWCNTs and b) spray deposited SWCNTs treated with OP. In both spectra, the peaks at 2916 and 2849 cm −1 could be assigned to C-H stretching vibrations, while C=C bending vibrations are observed at 983 and 831 cm −1 [2]. A broad peak at about 3460 cm −1 could be assigned to the O-H stretch, while the peak at 1217 cm −1 can be associated with C-O stretching vibrations, the intensity of the peaks is much higher after OP treatment which indicates the OP treatment led to the graft of polar groups [3]. Moreover, the peak at 1472 cm −1 can be associated with the C-O-H stretching [2]. FTIR results indicate the presence of O-H, C-O stretching vibrations rather than carbonyl groups could be the consequence of different initial surface conditions and Different OP parameters. Figure S3. Fourier-transform infrared spectra of (a) spray deposited SWCNTs and (b) spray deposited SWCNTs treated with OP.

Flexibility, Regeneration and Time Stability of Histamine Immunosensor
After the first regeneration and 100 bending cycles the current generation was 100.71% with a standard deviation of ±0.60 µA. After the second regeneration and 250 bending cycles the current generation was 94.66% with a standard deviation of ±0.78 µA. After third regeneration and 500 bending cycles the current generation was 77.97% with a standard deviation of ±0.96 µA. After the fourth generation and 1000 bending cycles the current generation was 52.85% with a standard deviation of ±2.51 µA. Every 2 days three sensors already prepared were tested to quantify 5 ng/mL of His diluted-on PBS pH = 7.4 for one month. After 12 days, the current generation was 95.29% of its initial value (as displayed in Figure S4).