An amperometric glucose biosensor prototype fabricated by thermal inkjet printing

Three-dimensional (3D) bioprinting techniques, developed over the past two decades, have the potential to fabricate living, patient-specific tissues and organs for use in regenerative medicine (Kesti et al. 2016). Inkjet-based bioprinting is one of the oldest yet promising technologies, which combines the solid freeform fabrication process and precise cell placement in 2D and 3D. Typically, inkjet printing takes digital data of an image or character from a computer and reproduces it on to a material or substrate using ink drops in a noncontact mode (Mohebi and Evans 2002). In addition to its wide application in automation office tool, it has also been widely used in microengineering for printing electronic materials (Sirringhaus et al. 2000). Inkjet printer deposits controlled volume of ink in a layer-by-layer fashion to predefined locations on successive layers to form a 3D construct (Xu et al. 2008). Drop-on-demand (DOD) printers, a commonly used inkjet printers, are used for biological and nonbiological applications. The idea of printing of biological substances was introduced by Klebe after using HP thermal DOD inkjet printer to deposit collagen and fibronectin in 1988 (Klebe 1988). Later, Thomas Boland customized the thermal inkjet printer for printing living cells in a viable state (Wilson and Boland 2003). After this, scientists have made great progress in patterning molecules such as DNA, depositing proteins (Eric et al. 2009, Ilkhanizadeh et al. 2007, Miller et al. 2006), and printing tissues and organs by inkjet printing (Okamoto et al. 2000). The current focus of inkjet bioprinting is fabrication of 3D structures with clinically significant heights to extend its effectiveness in the direction of clinical applications. In this chapter, we discuss about the principle of inkjet bioprinting technology, types of inkjet printers in use, materials used for printing, and the critical parameters involved in this technique. Then, challenges associated with techniques and the future perspectives are also discussed.

Enzyme-mediated amperometric biosensors prepared via successive surface-initiated atom-transfer radical polymerization

Amperometric glucose biosensor based on screen-printed carbon electrodes mediated with hexacyanoferrate–chitosan oligomers mixture

Hypothermia is a medical condition in which the human body’s core temperature is 35°C or lower, well below the nominal temperature of around 37°C. Accidental/unintentional hypothermia is generally divided into mild, moderate and severe. Severe hypothermia patients are re-warmed using active internal/external methods. There is a market for portable non-electric based warming blanket in pre-hospital treatment but such product is not currently available. In this project, feasibility of glucose oxidase (GOX) based reaction is examined for use in an active warming blanket. GOX was chosen because the enzyme and its substrates are readily available, relatively cheap, safe and have relatively long shelf-life; essential for subsequent commercialization. In addition, GOX from Aspergillus niger is a well characterised glycoprotein, its catalyzed reaction: the oxidation of D-glucose (C6H12O6) to D-gluconolactone (C6H10O6) and hydrogen peroxide, releases significant amount of energy (200 kJ mol-1) which is necessary for the application examined here. GOX is Generally Regarded As Safe (GRAS) and is the basis of many industrial applications. In depth study was conducted to understand the various fundamental properties which govern the glucose oxidase catalyzed enzymatic reaction. The result shows that there were two pH optimums as well as increasing temperature optimum at decreasing pH. Thermostability of GOX increases from pH 3.8 to 7.5 as well as in the presence of sodium chloride and/or glucose. Stabilization due to dissolved sodium chloride and glucose are cumulative because they act via different mechanism and was demonstrated with fluorescence emission. Upon drying, GOX activity decreases 60% immediately and gradually decreases further over longer time. Unexpectedly, additions of thermo-stabilizers do not increase the stability of dried GOX but actually accelerates its rate of degradation over time. These findings are important factors to consider when the proposed warming blanket utilizing GOX is ready for commercial use. To examine the heat transfer characteristics and performance of GOX reaction, a simple experimental model was built. The model consists of a reaction chamber where the reaction mixture is pumped into a prototype blanket insulated by polystyrene box. Fitting the experimental data into a hypothetical naked human model covered only with the prototype blanket, shows that the warming blanket concept utilizing the GOX heat generation reaction is feasible only if the reaction rate and/or scale of reaction is increased several times.

Aspergillus niger, is a filamentous fungus and producer of industrial enzymes such as glucose oxidases and cellulases. These enzymes are naturally produced by the fungus in order to digest and absorb nutrients from its environment. However, the enzyme quantities that are naturally produced are low. Thus, the aim of this study was to determine the effects of Psidum guajava leaf extract on the production of cellulases and glucose oxidases from Aspergillus niger. The leaves of Psidium guajava were extracted using absolute methanol. Aspergillus niger was then grown in the presence and absence of the extract in order to investigate the effects of extract on enzyme production by the fungus. The enzymes secreted into the broth medium were isolated by centrifugation. The supernatant which contained the secreted enzymes was used for the determination of enzyme activity. Enzyme activity was determined using the specific substrate for the specific enzyme, such as 2 % cellulose for cellulase and standard glucose solution for glucose oxidase. The results showed that the Psidium guajava leaf extract had an effect on production and activity of cellulases and glucose oxidases. From this study, it was noted that the Psidium guajava leaf extract may be used to induce the production of enzymes by Aspergillus niger and these enzymes may be of industrial use.

Interactions of ink colourants with chemically modified paper surfaces concerning inkjet print improvement

Thermal inkjet printing of thin-film electrolytes and buffering layers for solid oxide fuel cells with improved performance

Electrocatalytical oxidation of glucose on graphite using new mediators (1,6‐dithiapyrenes (DTPs) and 1,6‐dioxapyrenes) was performed. The electrocatalysis of glucose oxidase (GO) was based on the dithia(dioxa)pyrene cation‐radicals reaction with reduced GO. The rate of reduced GO oxidation by the cation‐radical of dithiapyrene was 4.8 × 104 M−1 s−1 at 25°C (pH 7.0). For enzyme electrode preparation, the GO was covalently immobilized by carbodiimide, and the mediator was adsorbed on a graphite electrode. The dithia(dioxa)pyrenes mediated glucose oxidation proceeds at potentials higher than 0.1 V (versus standard calomel electrode (SCE)). Of the investigated substituted dithia(dioxa)pyrenes, the largest electrocatalytical current was observed with electrodes comprising of DTP. Linear calibration graphs of these electrodes were obtained up to 12 mM of glucose, the maximal electrode activity was in the range of pH 6.5 to 8.0, and the temperature coefficient was 5.6%/°C. The investigated electrocatalytical systems can be used for construction of disposable senses for glucose determination in physiological solutions.

Modification of glucose oxidase for the development of biocatalytic solvent inks

Novel grafted electrochemical interface for covalent glucose oxidase immobilization using reactive pentafluorophenyl methacrylate.

The glucose oxidase gene (god) from Aspergillus niger was expressed in Hansenula polymorpha using the methanol oxidase promoter and transcription termination region and the MF-alpha leader sequence from Saccharomyces cerevisiae to direct secretion. The expression cassette was cloned into the S. cerevisiae vector YEp13 and used to transform H. polymorpha strain A16. In the initial transformants plasmid replication was unstable, but was stabilized by a growth regime consisting of alternating cycles of selective and non-selective growth. The stabilized strain was grown to high cell density by fed-batch fermentation. Upon induction of the MOX promoter, glucose oxidase synthesis was initiated. At the end of the fermentation, the culture density was 76 g dry weight/1 and 108 IU/ml (0.5 g/1 or 0.65% dry weight) glucose oxidase was found in the culture medium; a further 86 IU/ml (0.43 g/1 or 0.56% dry weight) was recovered from the cell lysate. A plate assay was used to monitor glucose oxidase levels in individual colonies. This was then used to isolate mutants which showed abnormal regulation of god expression or which showed an altered pattern of secretion. One mutant, which showed increased production of glucose oxidase, was grown to high cell density by fed-batch fermentation (100.6 g/l) and produced 445 IU/ml(2.25 g/l or 2.2% dry weight) extracellularly and 76 IU/ml (0.38 g/l or 0.4% dry weight) intracellularly. The mutant thus not only increased total production but exported 83% of the total enzyme made compared to 55% in the parent strain.

Modification of carbon and gold electrodes by a biocomposite film based on silica, hemoglobin (Hb), and glucose oxidase (GOx) has been performed by electro-assisted deposition using an electrogenerated catalyst. Proteins immobilized in the film retain catalytic activity for three weeks. The stability of the analytical signal of a modified electrode significantly increases on the addition of Au nanoparticles of the diameter 20 nm and cetyltrimethylammonium bromide (20 CMC) to the sol. A gold electrode modified with a biocomposite SiO2–Hb–GOx–Au film has been used for the voltammetric determination of glucose in model solutions and porcine blood serum. The calibration curve is linear in the range 0.015–0.20 mM; the limit of detection is 0.008 mM of glucose. The developed method of modification offers promise for the preparation of sensing elements of two-enzyme amperometric biosensors.

In order to develop better and alternative sintering methods, a basic understanding of thermal sintering and how particles sinter has been described. The conductivity development of commercially available silver inks has been discussed. Hereto, the resistance was measured on-line during the heating of the silver tracks from room temperature to 650 °C. The standard method, however, for sintering is by applying heat, typically above 200 °C, which is not compatible with the common polymer foils that are being considered as substrates for plastic electronics applications. By using microwave radiation instead of conventional radiation-conduction-convection heating, the sintering time of silver nanoparticles was shortened by a factor of 20, i.e. instead of 60 minutes three minutes were sufficient for sintering. The polymer substrate is virtually transparent to microwave radiation, whereas the conductive silver nanoparticles absorb the microwaves strongly and sinter. Furthermore, the presence of conductive antennae promotes nanoparticle sintering in precured ink lines to an extent that depends on the total area of the antennae. For cured nanoparticle inks that are connected to antennae, sintering times of 1 second are sufficient to obtain pronounced nanoparticle sintering. The antenna effect is greater if the ink line already exhibits conductivity. It is believed that this is due to the decreased mismatch between the impedance of air and sample. Using metal antennae, it was revealed that 1 second is sufficient to obtain pronounced sintering by microwave heating. The degree of sintering for these short exposure periods, however, strongly depends on the initial resistance of the pre-cured ink lines. After microwave flash sintering, the tracks revealed conductivity values of 10 to 34% compared to the bulk silver value, which is significantly higher compared to conventional heating methods.

Personalisation of warfarin therapy using thermal ink-jet printing

One of the major problems in amperometric biosensors based on detection of H2O2 produced by enzymatic reaction between oxidase enzymes and substrate is the interference of redox active compounds such as ascorbic acid (AA), dopamine (DA) and uric acid (UA). To minimize these interferences, sodium bismuthate was used for the first time as an insoluble pre‐oxidant in the flow injection (FI) amperometric glucose biosensor at a Glucose oxidase (GOx) immobilized Pt/Pd bimetallic modified pre‐anodized pencil graphite electrode (p.PGE). In this context, these interfering compounds were injected into a flow injection analysis (FIA) system using an injector which was filled with NaBiO3. Thus, these interferents were converted into their redox inactive oxidized forms before reaching the electrode in the flow cell. While glucose was not influenced by the pre‐oxidant in the injector, the huge oxidation peak currents of the interferents decreased significantly in the biosensor. FI amperometric current time curves showed that the AA, DA and UA were minimized by 96 %, 86 %, and 98 % respectively, in the presence of an equivalent concentration of interferences in a 1.0 mM glucose solution. The proposed FI amperometric glucose biosensor exhibits a wide linear range (0.01–10 mM, R2=0.9994) with a detection limit of 2.4×10−3 mM. Glucose levels in the artificial serum and two real samples were successfully determined using the fabricated FI amperometric biosensor.