"Stable-on-the-Table" Biosensors: Hemoglobin-Poly (Acrylic Acid) Nanogel BioElectrodes with High Thermal Stability and Enhanced Electroactivity

Abstract

In our efforts toward producing environmentally responsible but highly stable bioelectrodes with high electroactivities, we report here a simple, inexpensive, autoclavable high sensitivity biosensor based on enzyme-polymer nanogels. Met-hemoglobin (Hb) is stabilized by wrapping it in high molecular weight poly(acrylic acid) (PAA, M(W) 450k), and the resulting nanogels abbreviated as Hb-PAA-450k, withstood exposure to high temperatures for extended periods under steam sterilization conditions (122 °C, 10 min, 17-20 psi) without loss of Hb structure or its peroxidase-like activities. The bioelectrodes prepared by coating Hb-PAA-450k nanogels on glassy carbon showed well-defined quasi-reversible redox peaks at -0.279 and -0.334 V in cyclic voltammetry (CV) and retained >95% electroactivity after storing for 14 days at room temperature. Similarly, the bioelectrode showed ~90% retention in electrochemical properties after autoclaving under steam sterilization conditions. The ultra stable bioelectrode was used to detect hydrogen peroxide and demonstrated an excellent detection limit of 0.5 μM, the best among the Hb-based electrochemical biosensors. This is the first electrochemical demonstration of steam-sterilizable, storable, modular bioelectrode that undergoes reversible-thermal denaturation and retains electroactivity for protein based electrochemical applications.

Keywords: biocatalysis; electrochemistry; hemoglobin; high temperature catalysis; polyacrylic acid; steam sterilization.

Scheme 1

Hemoglobin binding to PAA (Middle) and their corresponding cyclic voltammograms (Right) and TEM’s (Left). TEM image of Hb-PAA shows the formation of nanogels (also shown in circle).

Figure 1

Covalent conjugation of Hb with PAA shown by agarose gel and TEM’s. (A) Agarose gel of Hb and Hb-PAA conjugates (40 mM Tris acetate pH 6.5). Lane 1, Hb; lane 2, Hb/PAA-450k(1:0.8:1.5) physical mixture; lane 3, Hb-PAA-450k(1:0.8:1.5); lane 4, Hb/PAA-450k(1:0.3:1.5) physical mixture, and lane 5, Hb-PAA-450k(1:0.3:1.5). Hb migrated towards negative electrode (lane 1) and Hb-PAA migrated (lanes 3 and 5) towards the positive electrode due to the negatively charged carboxyl groups of PAA conjugated to Hb; (B) TEM image of Hb (C) TEM image of Hb-PAA-450k(1:0.8:1.5) showing nanogels (also shown in circle). All TEM’s are after staining with uranyl acetate.

Figure 2

(A) Absorbance spectra of Hb, Hb-PAA-450k(1:0.8:1.5) and Hb-PAA-450k(1:0.3:1.5); (B) Far UV CD spectra of Hb, Hb-PAA-450k(1:0.8:1.5) and Hb-PAA-450k(1:0.3:1.5); (C) Kinetic traces of Hb,. Hb, Hb-PAA-450k(1:0.8:1.5) and Hb-PAA-450k(1:0.3:1.5). All data collected in 10 mM phosphate buffer pH 7.4.

Figure 3

(A) Specific activities (compared to Hb, 100%) of Hb-PAA-450k(1:0.8:1.5), Hb-PAA-450k(1:0.3:1.5), and Hb-PAA-8k(1:100:1) at room temperature; (B) Lineweaver-Burk plots for peroxidase activities of Hb, Hb-PAA-450k(1:0.8:1.5) and Hb-PAA-450k(1:0.3:1.5) samples; (C) Comparison of specific activities of Hb (compared to Hb, 100%), Hb-PAA-450k(1:0.8:1.5), Hb-PAA-450k(1:0.3:1.5), and Hb-PAA-8k(1:100:1) before (red) and after (blue) steam sterilization (cooled for 24 h). Each bar is an average of three trials. All reactions were carried out in the presence of 1 µM protein and 1 mM H₂O₂ in phosphate buffer pH 7.4, at room temperature.

Figure 4

Improved stability: Cyclic voltammograms of Hb-PAA electrodes, (A) in pH 7.4 PBS (0.1 M) before heat treatment; (B) Hb-PAA-450k(1:0.8:1.5) electrode after steam sterilization (SS) with 2 h cooling (black) and 24 h cooling (red) in pH 7.4 PBS (0.1 M); (C) Stability of Hb-PAA-450k(1:0.8:1.5) electrodes at room temperature over 14 days.

Figure 5

(A) Cyclic Volatmmograms of Hb-PAA-450k(1:0.8:1.5) modified GC electrode at different concentration of H₂O₂ (Inset: corresponding calibration plot); (B) Amperometric response of Hb-PAA modified electrode with successive additions of H₂O₂ in PB pH 7.4 at an applied potential of −0.335 V at 25 °C; (C) Corresponding calibration plot of amperometric response towards H₂O₂.