RESUMO
Drop-cast crosslinked hydrogels are a common platform for enzymatic electrochemical biosensors. Despite the widespread use of these complex systems, there are still several questions about how their physicochemical properties affect their performance, stability, and reproducibility. In this work, first-generation faradaic biosensors composed of glucose oxidase and branched polyethyleneimine (BPEI) are prepared using either glutaraldehyde (GA) or ethylene glycol diglycidyl ether (EGDGE) as crosslinkers. While EGDGE gels present an increasing electrochemical response with increasing crosslinker concentration, the current of GA gels decreases at high crosslinker concentration probably due to the hampered diffusion on tightly networked gels. We compared different strategies to use fluorescence microscopy to gain insight into the gel structure either by labeling the gel components with fluorophores or taking advantage of the intrinsic fluorescence of the imines formed upon crosslinking with GA. By monitoring the fluorescence of the crosslinking bonds and the electrochemical response, we demonstrate that hydrolysis, a common hydrogel degradation mechanism, is not responsible for the loss of electrical current over time in gels prepared with glutaraldehyde. Most hydrogel-based electrochemical biosensor studies do not perform specific experiments to determine the cause of the degradation and instead just infer it from the dependence of the current on the preparation conditions (most commonly concentrations). We show that, by taking advantage of several analytical techniques, it is possible to gain more knowledge about the degradation mechanisms and design better enzymatic biosensors.
RESUMO
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.
Assuntos
Técnicas Biossensoriais , Glucose , Glucose/metabolismo , Microfluídica , Polímeros/química , Peróxido de Hidrogênio , Glucose Oxidase/química , Oxirredução , Eletrodos , Técnicas de Cultura de Células em Três Dimensões , Técnicas Eletroquímicas , Enzimas Imobilizadas/químicaRESUMO
In situ examination of the reductive desorption process for Au microelectrodes modified with a thiol self-assembled monolayer (SAM) using fluorescence microscopy enabled the study of the fate of the desorbed thiolate species. The Bodipy labeled alkyl-thiol SAM, when adsorbed, is not fluorescent due to quenching by the Au surface. Once reductively desorbed, the thiolate molecules fluoresce and their direction and speed are monitored. At moderately negative reduction potentials, the thiolate species hemispherically diffuse away from the microelectrode. Also observed is the influence of a closely positioned counter electrode on the direction of the desorbed thiolate movement. As the potential becomes more negative, the molecules move in an upward direction, with a speed that depends on the amount of dissolved H(2) produced by water reduction. Shown is that this motion is controlled, in large part, by the change in the electrolyte density near the electrode due to dissolved H(2). These results should help in explaining the extent of readsorption at oxidative potentials observed in cyclic voltammetry (CV) reductive desorption measurements, as well as improving the general understanding of the SAM removal process by reductive desorption. The electrogenerated H(2) was also shown to be able to reductively remove the thiol SAM from the Pt/Ir particles that decorate the microelectrode glass sheath.