Development of a Redox-Polymer-Based Electrochemical Glucose Biosensor Suitable for Integration in Microfluidic 3D Cell Culture Systems

Abstract

The inclusion of online, in situ biosensors in microfluidic cell cultures is important to monitor and characterize a physiologically mimicking environment. This work presents the performance of second-generation electrochemical enzymatic biosensors to detect glucose in cell culture media. Glutaraldehyde and ethylene glycol diglycidyl ether (EGDGE) were tested as cross-linkers to immobilize glucose oxidase and an osmium-modified redox polymer on the surface of carbon electrodes. Tests employing screen printed electrodes showed adequate performance in a Roswell Park Memorial Institute (RPMI-1640) media spiked with fetal bovine serum (FBS). Comparable first-generation sensors were shown to be heavily affected by complex biological media. This difference is explained in terms of the respective charge transfer mechanisms. Under the tested conditions, electron hopping between Os redox centers was less vulnerable than H₂O₂ diffusion to biofouling by the substances present in the cell culture matrix. By employing pencil leads as electrodes, the incorporation of these electrodes in a polydimethylsiloxane (PDMS) microfluidic channel was achieved simply and at a low cost. Under flow conditions, electrodes fabricated using EGDGE presented the best performance with a limit of detection of 0.5 mM, a linear range up to 10 mM, and a sensitivity of 4.69 μA mM^-1 cm^-2.

Keywords: 3D cell culture; EGDGE; cross-linking; glucose electrochemical biosensor; glucose oxidase; glutaraldehyde; on-chip evaluation.

Figure 1

Schematic of the microfluidic channel with the integrated carbon pencil leads as working (WE) and counter (CE) electrodes and a Ag|AgCl wire as pseudo-reference electrode (RE).

Figure 2

(A,C) Cyclic voltammograms of hydrogels containing glucose oxidase (GOx) cross-linked to branched polyethyleneimine modified with Os(bpy)₂Cl(pyCOH) (OsBPEI) using either glutaraldehyde (GA) or ethylene glycol diglycidyl ether (EGDGE). (A) OsBPEI/GOx/GA and (C) OsBPEI/GOx/EGDGE hydrogels with different cross-linker concentrations in 0.1 M pH 7.4 phosphate buffer (PB), with 0 mM (dotted lines) and 100 mM (continuous lines) glucose. Scan rate: 2 mV/s. (B,D) Calibration curves for 0–100 mM glucose in0.1 M pH 7.4 PB, based on chronoamperometric evaluations using (B) OsBPEI/GOx/GA and (D) OsBPEI/GOx/EGDGE hydrogels with different cross-linker concentrations. Error bars represent the standard deviation between three independent electrodes. Dashed lines represent the corresponding curves employing the apparent Michaelis–Menten constant (K_m^app) and maximum current (i_max) calculated through non-linear fitting.

Figure 3

(A) Glucose calibration curves (0–100 mM) in Roswell Park Memorial Institute (RPMI-1640) culture medium with OsBPEI/GOx/GA or OsBPEI/GOx/EGDGE 33.3 mM hydrogels. Error bars represent the standard deviation of three independent electrodes. Dashed lines represent the corresponding curves employing the K_m^app and i_max calculated through non-linear fitting. (B) Evaluation of OsBPEI/GOx/GA or OsBPEI/GOx/EGDGE 33.3 mM hydrogels with concentrations of fetal bovine serum (FBS) between 0–10% in RPMI-1640 culture medium containing 5 mM glucose.

Figure 4

(A,B) Calibration curves of 0–100 mM glucose in (A) 0.1 M pH 7.4 PB and (B) RPMI-1640 culture medium, based on chronoamperometric evaluations employing BPEI/GOx/GA or BPEI/GOx/EGDGE first-generation biosensors. Error bars represent the standard deviation of three independent electrodes. Dashed lines represent the corresponding curves employing the K_m^app and i_max calculated through non-linear fitting. The first point (blank) in the EGDGE series in (A) was not considered for the fitting. (C,D) Schematics of the effects of adsorption and absorption of media components on the charge transfer mechanisms of first- (C) and second- (D) generation biosensors.

Figure 5

(A–C) Bright-field (A) fluorescence (B) and scanning electron microscopy (C) images of a Foray HB pencil lead electrode with a deposit of OsBPEI/GOx/GA hydrogel. (D–G) Energy dispersive X-ray (EDX) mapping images of the same electrode corresponding to carbon (D), oxygen (E), osmium (F), and phosphorus (G). All scale bars are 500 μm in length. (H) Chronoamperometric evaluations of the modified electrode in 0.1 M pH 7.4 PB, RPMI-1640 culture medium, and DMEM culture medium with glucose concentrations between 0–25 mM.

Figure 6

(A) Chronoamperometric calibration curves of 0–100 mM glucose in RPMI-1640 culture medium and 5% FBS employing pencil lead electrodes modified with OsBPEI/GOx/GA or OsBPEI/GOx/EGDGE in the microfluidic channel. Error bars represent the standard deviation of three different electrodes. Dashed lines represent the corresponding curves employing the K_m^app and i_max calculated through non-linear fitting. (B) Photograph of the microfluidic channel with the integrated electrodes. Red dye has been injected in the channel to aid in its visualization.