A Third Generation Glucose Biosensor Based on Cellobiose Dehydrogenase Immobilized on a Glassy Carbon Electrode Decorated with Electrodeposited Gold Nanoparticles: Characterization and Application in Human Saliva

Abstract

Efficient direct electron transfer (DET) between a cellobiose dehydrogenase mutant from Corynascus thermophilus (CtCDH C291Y) and a novel glassy carbon (GC)-modified electrode, obtained by direct electrodeposition of gold nanoparticles (AuNPs) was realized. The electrode was further modified with a mixed self-assembled monolayer of 4-aminothiophenol (4-APh) and 4-mercaptobenzoic acid (4-MBA), by using glutaraldehyde (GA) as cross-linking agent. The CtCDH C291Y/GA/4-APh,4-MBA/AuNPs/GC platform showed an apparent heterogeneous electron transfer rate constant (ks) of 19.4 ± 0.6 s-1, with an enhanced theoretical and real enzyme surface coverage (Γtheor and Γreal) of 5287 ± 152 pmol cm-2 and 27 ± 2 pmol cm-2, respectively. The modified electrode was successively used as glucose biosensor exhibiting a detection limit of 6.2 μM, an extended linear range from 0.02 to 30 mM, a sensitivity of 3.1 ± 0.1 μA mM-1 cm-2 (R2 = 0.995), excellent stability and good selectivity. These performances compared favourably with other glucose biosensors reported in the literature. Finally, the biosensor was tested to quantify the glucose content in human saliva samples with successful results in terms of both recovery and correlation with glucose blood levels, allowing further considerations on the development of non-invasive glucose monitoring devices.

Keywords: cellobiose dehydrogenase; electrodeposition; glucose biosensor; gold nanoparticles; human saliv.

Scheme 1

The electrode modification pathway for CtCDH C291Y/GA/4-APh,4-MBA/AuNPs/GC platform has been reported. Initially, AuNPs were directly electrodeposited onto cleaned GC electrode by sweeping the potential. Afterward, the electrode was incubated in a thiol mixture (1:1 v/v 4-APh and 4-MBA) overnight, followed by cross-linking reaction (glutaraldehyde GA: cross-linking agent) to covalently link the enzyme (CtCDH C291Y) to the so modified electrode surface.

Figure 1

SEM images of AuNPs-modified GCEs obtained by sweeping the potential between 1.1 and −0.1 V vs. Ag|AgCl_sat for a given number of scans in 10 mM HAuCl₄ (0.5 M H₂SO₄): (a) 5, (b) 10, (c) 15, (d) 20, (e) 25, (f) 30 and (g) 35.

Figure 2

Cyclic voltammograms of AuNPs modified GCE electrodes with different number of scans (from 0 scans to 35 scans) in 0.5 M H₂SO₄. Scan rate 100 mV s⁻¹. T = 25 °C.

Figure 3

(a) CVs of CtCDH C291Y/GA/4-APh,4-MBA/AuNPs/GC in pH 7.4, 50 mM TRIS (0.1 M KCl) in the potential range of −0.6 V to 0.35 V, scan rates from 2 mV s⁻¹ to 500 mV s⁻¹. Inset: linear part of cathodic (J_pc) and anodic peak current densities (J_pa) vs. scan rate (v). (b) CVs of CtCDH C291Y/GA/4-APh, 4-MBA/AuNPs/GC in absence (black) and in presence of 5 mM glucose (red) in 50 mM TRIS buffer, pH 7.4 (0.1 M KCl) at a scan rate of 5 mV s⁻¹. T = 25 °C.

Figure 4

(a) Glucose biosensor calibration graph of CtCDH C291Y/GA/4-APh,4-MBA/AuNPs/GC in 50 TRIS buffer pH 7.4 (0.1 M KCl); E_app = 0.250 vs. Ag|AgCl_sat; flow rate 0.5 mL min⁻¹; inset: linear part of the calibration curve; (b) biosensor response over the pH range (4.5–8): 50 mM acetate buffer (black), 50 mM MOPS buffer (red) and in 50 mM TRIS buffer (blue), with E_app = 0.250 vs. Ag|AgCl_sat; flow rate 0.5 mL min⁻¹; (c) biosensor response over the T range (20–45 °C) in TRIS buffer; (d) lifetime of CtCDH C291Y/GA/4-APh,4-MBA/AuNPs/GC biosensor in presence of 750 μM glucose solution; (e) influence of interfering compounds on glucose response in presence of 750 μM glucose, maltose, cortisol, ascorbic acid, Ca²⁺. Experimental conditions (c–e): 50 TRIS buffer pH 7.4 (0.1 M KCl), E_app = 0.250 vs. Ag|AgCl_sat; flow rate 0.5 mL min⁻¹; injection volume 50 μL; T = 25 °C.

Figure 5

Correlation between glucose blood and saliva glucose concentration in 6 healthy patients at fasting state. Salivary glucose was detected with the CtCDH C291Y/GA/4-APh,4-MBA/AuNPs/GC biosensor, blood glucose was detected with the commercial GlucoContour XT.