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A graphene-functionalized carbon fiber electrode was modified with adsorbed polyethylenimine to introduce amino functionalities and then with trigonelline and 4-carboxyphenylboronic acid covalently bound to the amino groups. The trigonelline species containing quarterized pyridine groups produced positive charge on the electrode surface regardless of the pH value, while the phenylboronic acid species were neutral below pH 8 and negatively charged above pH 9 (note that their pKa=8.4). The total charge on the monolayer-modified electrode was positive at the neutral pH and negative at pH > 9. Note that 4-carboxyphenylboronic acid was attached to the electrode surface in molar excess to trigonelline, thus allowing the negative charge to dominate on the electrode surface at basic pH. Negatively charged fluorescent dye-labeled insulin (insulin-FITC) was loaded on the modified electrode surface at pH 7.0 due to its electrostatic attraction to the positively charged interface. The local pH in close vicinity to the electrode surface was increased to ca. 9–10 due to consumption of H+ ions upon electrochemical reduction of oxygen proceeding at the potential of −1.0 V (vs. Ag/AgCl) applied on the modified electrode. The process resulted in recharging of the electrode surface to the negative value due to the formation of the negative charge on the phenylboronic acid groups, thus resulting in the electrostatic repulsion of insulin-FITC and stimulating its release from the electrode surface. The insulin release was characterized by fluorescence spectroscopy (using the FITC-labeled insulin), by electrochemical measurements on an iridium oxide, IrOx, electrode and by mass spectrometry. The graphene-functionalized carbon fiber electrode demonstrated significant advantages in the signal-stimulated insulin release comparing with the carbon fiber electrode without the graphene species.
Prior to immobilization of biomolecules or cells onto biosensor surfaces, the surface must be physically or chemically activated for further functionalization. Organosilanes are a versatile option as they facilitate the immobilization through their terminal groups and also display self-assembly. Incorporating hydroxyl groups is one of the important methods for primary immobilization. This can be done, for example, with oxygen plasma treatment. However, this treatment can affect the performance of the biosensors and this effect is not quite well understood for surface functionalization. In this work, the effect of O2 plasma treatment on EIS sensors was investigated by means of electrochemical characterizations: capacitance–voltage (C–V) and constant capacitance (ConCap) measurements. After O2 plasma treatment, the potential of the EIS sensor dramatically shifts to a more negative value. This was successfully reset by using an annealing process.
An array of four independently wired indium tin oxide (ITO) electrodes was used for electrochemically stimulated DNA release and activation of DNA-based Identity, AND and XOR logic gates. Single-stranded DNA molecules were loaded on the mixed poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA)/poly(methacrylic acid) (PMAA) brush covalently attached to the ITO electrodes. The DNA deposition was performed at pH 5.0 when the polymer brush is positively charged due to protonation of tertiary amino groups in PDMAEMA, thus resulting in electrostatic attraction of the negatively charged DNA. By applying electrolysis at −1.0 V(vs. Ag/AgCl reference) electrochemical oxygen reduction resulted in the consumption of hydrogen ions and local pH increase near the electrode surface. The process resulted in recharging the polymer brush to the negative state due to dissociation of carboxylic groups of PMAA, thus repulsing the negatively charged DNA and releasing it from the electrode surface. The DNA release was performed in various combinations from different electrodes in the array assembly. The released DNA operated as input signals for activation of the Boolean logic gates. The developed system represents a step forward in DNA computing, combining for the first time DNA chemical processes with electronic input signals.
A light-addressable potentiometric sensor (LAPS) is a field-effect-based potentiometric sensor with an electrolyte/insulator/semiconductor (EIS) structure, which is able to monitor analyte concentrations of (bio-)chemical species in aqueous solutions in a spatially resolved way. Therefore, it is also an appropriate tool to record 2D-chemical images of concentration variations on the sensor surface. In the present work, two differential, LAPS-based measurement principles are introduced to determine the metabolic activity of Escherichia coli (E. coli) K12 and Chinese hamster ovary (CHO) cells as test microorganisms. Hereby, we focus on i) the determination of the extracellular acidification rate (ΔpH/min) after adding glucose solutions to the cell suspensions; and ii) recording the amplitude increase of the photocurrent (Iph) related to the produced acids from E. coli K12 bacteria and CHO cells on the sensor surface by 2D-chemical imaging. For this purpose, 3D-printed multi-chamber structures were developed and mounted on the planar sensor-chip surface to define four independent compartments, enabling differential measurements with varying cell concentrations. The differential concept allows eliminating unwanted drift effects and, with the four-chamber structures, measurements on the different cell concentrations were performed simultaneously, thus reducing also the overall measuring time.
Three amperometric biosensors have been developed for the detection of L-malic acid, fumaric acid, and L -aspartic acid, all based on the combination of a malate-specific dehydrogenase (MDH, EC 1.1.1.37) and diaphorase (DIA, EC 1.8.1.4). The stepwise expansion of the malate platform with the enzymes fumarate hydratase (FH, EC 4.2.1.2) and aspartate ammonia-lyase (ASPA, EC 4.3.1.1) resulted in multi-enzyme reaction cascades and, thus, augmentation of the substrate spectrum of the sensors. Electrochemical measurements were carried out in presence of the cofactor β-nicotinamide adenine dinucleotide (NAD+) and the redox mediator hexacyanoferrate (III) (HCFIII). The amperometric detection is mediated by oxidation of hexacyanoferrate (II) (HCFII) at an applied potential of + 0.3 V vs. Ag/AgCl. For each biosensor, optimum working conditions were defined by adjustment of cofactor concentrations, buffer pH, and immobilization procedure. Under these improved conditions, amperometric responses were linear up to 3.0 mM for L-malate and fumarate, respectively, with a corresponding sensitivity of 0.7 μA mM−1 (L-malate biosensor) and 0.4 μA mM−1 (fumarate biosensor). The L-aspartate detection system displayed a linear range of 1.0–10.0 mM with a sensitivity of 0.09 μA mM−1. The sensor characteristics suggest that the developed platform provides a promising method for the detection and differentiation of the three substrates.