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. 2022 Sep 26:9:972008.
doi: 10.3389/fmolb.2022.972008. eCollection 2022.

Nanocomposite films as electrochemical sensors for detection of catalase activity

Dwight Johnson  1 , Unyoung Kim  1 , Maryam Mobed-Miremadi  1
Affiliations

Nanocomposite films as electrochemical sensors for detection of catalase activity

Dwight Johnson et al. Front Mol Biosci. .

Abstract

Cross-linked hydrogel substrates have garnered attention as they simultaneously enable oxidoreductase reactions in a control volume extended to adsorption of redox capacitors for amplification of electrochemical signals. In this study, the effect of catalase immobilization in mold-casted alginate-based thin films (1 mm × 6 mm × 10 mm) containing multi walled carbon nanotubes (MWCNT) coated with chitosan has been studied via amperometry. The amperometric response was measured as a function of peroxide concentration, at a fixed potential of -0.4 V vs. SPCE in phosphate-buffered saline (pH = 7.4). Results indicate substrate detection is not diffusion-limited by the 100 μm thick chitosan layer, if the cationic polyelectrolyte is in contact with the sensing carbon electrode, and the linear detection of the enzyme absent in solution is enabled by immobilization (R 2 = 0.9615). The ferricyanide-mediated biosensor exhibited a sensitivity of 4.55 μA/mM for the optimal formulation at room temperature comparable to other nanomaterial hybrid sensing solution namely amine-functionalized graphene with an average response time of 5 s for the optimal formulation. The suitability of the optimized chitosan-coated alginate slabs nano-environment for co-encapsulation of catalase and carbon nanotubes was confirmed by cyclic voltammetry.

Keywords: CNT; alginate; biosensor; catalase; chitosan; electrochemical; encapsulation; nanocomposite.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Redox reactions and associated mediators for the electrochemical detection of catalase (A) (left) Oxidation of hexacyanoferrate (II) by HRP, (B) (right) reduction of hexacyanoferrate (III) by H2O2. Molecular structure of the enzymes obtained from the protein database (Berglund et al., 2022; Sugadev et al., 2022).
FIGURE 2
FIGURE 2
Surface coating of nanocomposites (A) Alginate control film containing SWCNT, (B) Chitosan coated alginate film containing SWCNT with a 100 μm added thickness captured at ×40 magnification; (C) Nanocomposite film prior (translucent) and post (translucent green) electrochemical processing; (D) Screen-printed sensor with carbon working, silver reference, carbon counter electrodes and nanocomposite film used for H2O2.
FIGURE 3
FIGURE 3
(A) (top) Superimposed sample amperograms for Free HRP and 8 mM of H2O2 recorded at multiple voltages for a total scan time of 10 s using which the steady state sampling times were estimated. (B) (bottom) Outset dashed magnified region for the determination of the maximum current (Imax@S) used for the signal to noise estimations.
FIGURE 4
FIGURE 4
Voltage vs. current based on amperometric data for the estimation of the signal to noise ratio. (A) Free enzyme; (B) RSHRP nanocomposite film where chitosan is not in contact with the SPCE; (C) SHRP nanocomposite film where chitosan is in contact with the SPCE; (D) Free enzyme denoted as FreeCAT; (E) RSCAT nanocomposite film where chitosan is not in contact with the SPCE; (F) SCAT nanocomposite film where chitosan is in contact with the SPCE.
FIGURE 5
FIGURE 5
Amperometric studies of horseradish peroxidase measured at −0.1 V using H2O2 as substrate and corresponding layer stacking configurations (not drawn to scale). (A) Free enzyme; (B) RSHRP nanocomposite film where chitosan is not in contact with the SPCE; (C) SHRP nanocomposite film where chitosan is in contact with the SPCE.
FIGURE 6
FIGURE 6
Amperometric studies of catalase measured at -0.4 V using H2O2 as substrate and corresponding layer stacking configurations (not drawn to scale). (A) Free enzyme denoted as FreeCAT; (B) RSCAT nanocomposite film where chitosan is not in contact with the SPCE; (C) SCAT nanocomposite film where chitosan is in contact with the SPCE.
FIGURE 7
FIGURE 7
Voltammograms of nanocomposite biosensing films performed with a voltage sweep between 0.5 V and −0.8 V at a rate of 100 mV/s on controls and corresponding reactive catalase immobilized slabs in the top and bottom rows, respectively. (A) alginate slabs; (B) alginate slabs with CNTs (A/CNT); (C) alginate slabs with CNTs and chitosan (A/CNT/Chi); (D) alginate slabs with CAT (AE); (E) alginate slabs with CNTs and CAT (A/CNT + E); (F) alginate slabs with CNTs, chitosan and CAT (A/CNT/Chi + E).
FIGURE 8
FIGURE 8
Peak current at −0.4 V relative to the background signal from the voltagrams of nanocomposite biosensing films, performed with a voltage sweep between 0.5 V and −0.8 V at a rate of 100 mV/s on controls and corresponding reactive catalase immobilized slabs in the top and bottom rows, respectively. Main plots capture substrate ranging from (0–3 µL) while insets contrast the overlay between (0and3 µL): (A1) alginate slabs (A), (A2, inset); (B) alginate slabs with CNTs (A/CNT), (B2, inset); (C) alginate slabs with CNTs and chitosan (A/CNT/Chi), (C2,inset); (D) alginate slabs with CAT (AE), (D2,inset); (E) alginate slabs with CNTs and CAT (A/CNT + E), (E2, inset); (F) alginate slabs with CNTs, chitosan and CAT (A/CNT/Chi + E), (F2,inset).
FIGURE 9
FIGURE 9
Peak current at −0.4 V relative to the background signal from the voltammograms of nanocomposite biosensing films for 2 μl of H2O2 (8 mM).
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