Ratiometric Fluorescent Biosensors for Glucose and Lactate Using an Oxygen-Sensing Membrane

Abstract

In this study, ratiometric fluorescent glucose and lactate biosensors were developed using a ratiometric fluorescent oxygen-sensing membrane immobilized with glucose oxidase (GOD) or lactate oxidase (LOX). Herein, the ratiometric fluorescent oxygen-sensing membrane was fabricated with the ratio of two emission wavelengths of platinum meso-tetra (pentafluorophenyl) porphyrin (PtP) doped in polystyrene particles and coumarin 6 (C6) captured into silica particles. The operation mechanism of the sensing membranes was based on (i) the fluorescence quenching effect of the PtP dye by oxygen molecules, and (ii) the consumption of oxygen levels in the glucose or lactate oxidation reactions under the catalysis of GOD or LOX. The ratiometric fluorescent glucose-sensing membrane showed high sensitivity to glucose in the range of 0.1-2 mM, with a limit of detection (LOD) of 0.031 mM, whereas the ratiometric fluorescent lactate-sensing membrane showed the linear detection range of 0.1-0.8 mM, with an LOD of 0.06 mM. These sensing membranes also showed good selectivity, fast reversibility, and stability over long-term use. They were applied to detect glucose and lactate in artificial human serum, and they provided reliable measurement results.

Keywords: coumarin 6; glucose oxidase; glucose sensor; lactate oxidase; lactate sensor; porphyrin dye; ratiometric fluorescent sensor.

Scheme 1

Layout of a ratiometric fluorescent biosensor.

Figure 1

(a) TEM and fluorescence images of PS@PtP with a scale bar of 1 µm and Si@C6 with a scale bar of 200 nm; (b) 2D-fluorescence spectra of the PS@PtP*Si@C6 membrane to 0% and 100% oxygen gas.

Figure 1

(a) TEM and fluorescence images of PS@PtP with a scale bar of 1 µm and Si@C6 with a scale bar of 200 nm; (b) 2D-fluorescence spectra of the PS@PtP*Si@C6 membrane to 0% and 100% oxygen gas.

Figure 2

(a) SEM images of the EC membrane (left photos) and the PS@PtP*Si@C6 membranes (right photos) at different magnifications; (b) repeatability of the PS@PtP*Si@C6 membrane when exposed to concentrations of 100% and 0% oxygen gas; (c) photo images of the PS@PtP*Si@C6 membrane when exposed to different concentrations of oxygen gas.

Figure 2

(a) SEM images of the EC membrane (left photos) and the PS@PtP*Si@C6 membranes (right photos) at different magnifications; (b) repeatability of the PS@PtP*Si@C6 membrane when exposed to concentrations of 100% and 0% oxygen gas; (c) photo images of the PS@PtP*Si@C6 membrane when exposed to different concentrations of oxygen gas.

Scheme 2

(a) Reaction of epoxy group of GPTMS and amine group of enzyme (E); (b) reaction of amine group of APTMS with carboxyl group of enzyme (E).

Figure 3

(a) Immobilization efficiency of different amounts of GOD, and slope values of the calibration curves of the glucose-sensing membranes in the glucose concentration range from 0 to 2 mM; (b) immobilization efficiency of different amounts of LOX, and slope values of the calibration curves of the lactate-sensing membranes in the lactate concentration range from 0 to 0.8 mM; (c) SEM images of the oxygen-sensing membrane before and after LOX immobilization.

Figure 4

Response of the glucose-sensing membrane at different glucose concentrations, and calibration curve for glucose as determined by the ratio of FI₆₃₅/FI₄₇₅.

Figure 5

Reversibility of the glucose-sensing membrane repeatedly exposed at 0 and 2 mM glucose.

Figure 6

Response of the glucose-sensing membrane at different glucose concentrations in the temperature range of 25–35 °C and the pH range of 5–9.

Figure 7

(a) Response of the glucose-sensing membrane at 1 mM glucose (control sample), with and without the addition of 145 mM Na⁺, 106 mM Cl⁻, 30 mM HCO₃⁻, 1.625 mg/L Fe³⁺, and 5 g/dL BSA; (b) lifetime of the glucose-sensing membrane at different glucose concentrations.

Figure 8

Response of the lactate-sensing membrane at different lactate concentrations, and calibration curve for lactate as determined by ratio of FI₆₃₅/FI₄₇₅.

Figure 9

Reversibility of the lactate-sensing membrane repeatedly exposed at 0 and 1 mM lactate.

Figure 10

Response of the lactate-sensing membrane at different lactate concentrations in the temperature range of 25–35 °C and pH range of 5.0–9.0.

Figure 11

(a) Response of the lactate-sensing membrane at 1 mM lactate (control sample), with and without the addition of 145 mM Na⁺, 106 mM Cl⁻, 30 mM HCO₃⁻, 1.625 mg/L Fe³⁺, and 5 g/dL BSA; (b) lifetime of the lactate-sensing membrane at different lactate concentrations.

Figure 12

(a) Response of the glucose-sensing membrane to solutions of standard glucose and artificial human plasma glucose, and (b) response of the lactate-sensing membrane to solutions of standard lactate and artificial human plasma lactate. Percent difference of ratiometric fluorescence intensities (R = FI₆₃₅/FI₄₇₅) in glucose (or lactate) concentrations between standard solution and artificial human plasma represents 100 × {(R_{std glucose}-R_{human plasma glucose})/R_{std glucose}} or 100 × {(R_{std lactate}-R_{human plasma lactate})/R_{std lactate}}.