Flavocytochrome b2-Mediated Electroactive Nanoparticles for Developing Amperometric L-Lactate Biosensors

Abstract

L-Lactate is an indicator of food quality, so its monitoring is essential. Enzymes of L-Lactate metabolism are promising tools for this aim. We describe here some highly sensitive biosensors for L-Lactate determination which were developed using flavocytochrome b₂ (Fcb₂) as a bio-recognition element, and electroactive nanoparticles (NPs) for enzyme immobilization. The enzyme was isolated from cells of the thermotolerant yeast Ogataea polymorpha. The possibility of direct electron transfer from the reduced form of Fcb₂ to graphite electrodes has been confirmed, and the amplification of the electrochemical communication between the immobilized Fcb₂ and the electrode surface was demonstrated to be achieved using redox nanomediators, both bound and freely diffusing. The fabricated biosensors exhibited high sensitivity (up to 1436 A·M^-1·m^-2), fast responses, and low limits of detection. One of the most effective biosensors, which contained co-immobilized Fcb₂ and the hexacyanoferrate of gold, having a sensitivity of 253 A·M^-1·m^-2 without freely diffusing redox mediators, was used for L-Lactate analysis in samples of yogurts. A high correlation was observed between the values of analyte content determined using the biosensor and referenced enzymatic-chemical photometric methods. The developed biosensors based on Fcb₂-mediated electroactive nanoparticles can be promising for applications in laboratories of food control.

Keywords: L-Lactate; amperometric biosensor; electroactive nanoparticles; flavocytochrome b2.

Figure A1

Structural and morphological characterizations of the AuHCF/NPs (a,c) and PtZn/NPs (b,d); the SEM images (a,b) with the correspondent XRM spectra (c,d).

Figure A2

Properties of the Fcb_2-based ABSs. The selectivity test for AuHCF/GE (ABS-2); current outputs in relative units (%) on the added compounds, up to 2 mM concentration. As a 100% signal, the highest current response on L-Lact was taken (a). Stabilities of the NPs-based ABS-2, and control ABS-5 without NPs (b).

Figure A3

Assay of Lact in the samples of different yogurts (1–3) via graph SAT method (a–c, respectively), using Fcb₂/AuHCF/GE (25 mU Fcb₂ per ABS).

Figure A4

Correlation between the results of the determination of Lact content (mM) in drinking yogurts via reference enzymatic–chemical (Fcb₂/PB) and biosensor methods.

Figure 1

The principal scheme of Lact determination using the Fcb₂-based ABS.

Figure 2

Amperometric characteristics of the Fcb₂/GE as current responses to the increasing concentrations of Lact. (a) CV profiles at Lact concentrations: 0 mM (1, black), 2 mM (2, red), and 5 mM (3, blue); (b) chronoamperogram at the working potential of −75 mV.

Figure 3

CV profiles of the control GE (a–c, line 1) and the modified electrodes: AuHCF/GE (a, 2), PtZn/GE (b, 2) and gCuHCF/GE (c, 2). Conditions: 10 mM K₃Fe(CN)₆, 100 mM KCl in 50 mM phosphate buffer, pH 6.5, and 20 °C.

Figure 4

Calibration curves for Lact determination in wide (left) and linear (right) ranges using the Fcb₂/NPs/GEs: (a)control, without NPs; (b–f) samples with AuHCF (b), PdHCF (c), PtHCF (d), PtZn (e), and NiPtPd (f). Each bioelectrode contained 25 mU of Fcb₂ and 1 µg of NPs on the surface of the GE. Conditions: 50 mM phosphate buffer, pH 8.0, and working potential of −75 mV.

Figure 4

Calibration curves for Lact determination in wide (left) and linear (right) ranges using the Fcb₂/NPs/GEs: (a)control, without NPs; (b–f) samples with AuHCF (b), PdHCF (c), PtHCF (d), PtZn (e), and NiPtPd (f). Each bioelectrode contained 25 mU of Fcb₂ and 1 µg of NPs on the surface of the GE. Conditions: 50 mM phosphate buffer, pH 8.0, and working potential of −75 mV.

Figure 5

Calibration curves of the Fcb₂/AuHCF/GEs in the broad (a) and linear (b) ranges of the dependence of enzyme quantity on the surface of GE; lines 1, 2, 3 correspond to 13, 25 and 50 mU of Fcb₂, respectively. The working potential is −75 mV.

Figure 6

Impact of electroactive mediators, both immobilized (AuHCF and PtZn) and freely diffusing (PMS), on the sensitivities of the ABSs to Lact. ABSs with compositions Fcb₂/GE, Fcb₂/AuHCF/GE, and Fcb₂/PtZn/GE were tested without PMS (1, yellow columns) and in the presence of PMS (2, cyan columns) in a 50 mM phosphate buffer, at pH 8.0, and at a working potential of −75 mV.