Cheap and Sustainable Biosensor Fabrication by Enzyme Immobilization in Commercial Polyacrylic Acid/Carbon Nanotube Films.

Novel glucose biosensors were constructed by loading glucose oxidase (GOx) into the nanopores of homogenous carbon nanotube (CNT) films on the surface of Pt disk electrodes and trapping the enzyme by subsequent deposition of polyacrylic acid (PAA), forming PAA/GOx-CNT-modified Pt disks. In amperometric biosensing with anodic hydrogen peroxide (H2O2) detection at a potential of +600 mV, increasing electrolyte glucose concentrations produced instantaneous steps in the H2O2 oxidation current. Glucose biosensor amperometry was feasible down to 10 µM, with a sensitivity of about 34 µA mM-1 cm-2 and linear current response up to 5 mM. The biosensors reliably determined glucose concentrations in human serum and a beverage. Successful trials with PAA/GOx-CNT-modified screen-printed Pt electrode disks demonstrated the potential of this means of enzyme fixation in biosensor mass fabrication, which offers a unique combination of cheap availability of the two matrix constituents and sensor layer formation through simple drop-and-dry steps. PAA/GOx-CNT/Pt biosensors are green and user-friendly bioanalytical tools that do not need large budgets, special skills, or laboratory amenities for their production. Any user, from industrial, university, or school laboratories, even if inexperienced in biosensor construction, can prepare functional biosensors with GOx, as in these proof-of-principle studies, or with other redox enzymes, for clinical, environmental, pharmaceutical, or food sample analysis.

Prussian Blue/Carbon Nanotube Sensor Spread with Gelatin/Zein Glaze: A User-Friendly Modification for Stable Interference-Free H2O2 Amperometry.

We report the production and characterization of effective amperometric sensors for cathodic hydrogen peroxide (H2O2) detection. The proposed electrodes involve a combination of a H2O2-signaling Prussian Blue (PB)/carbon nanotube (CNT) layer with a glaze of the biopolymers gelatin (top) and zein (beneath) for protection against PB leakage. The sandwich-type sensor was constructed through simple "drop and dry" steps with (1) suspensions of the CNTs in a soluble PB solution, (2) zein in ethanol, and (3) gelatin in water, applied sequentially to the carbon working electrode disk of a screen-printed carbon electrode (SPCE) platform. The PB in the signaling layer acted as the electrocatalyst for H2O2 reduction at -150 mV vs Ag/AgCl/3 M KCl, enabling cathodic H2O2 amperometry with good target proportionality. Calibration trials confirmed the linearity of the response up to 700 µM (R2 > 0.998), with a sensitivity of 0.425 µA µM-1 cm-2 and a practical detection limit of 1 µM. Quantification of H2O2 in model and real samples with gelatin-zein-PB/CNT-SPCEs had a recovery of close to 100% of the true value. Since they are easily and cheaply made and yield accurate target assessments, gelatin-zein-PB/CNT-SPCEs are an ideal tool for electrochemical H2O2 analyses in human body fluids, health care products, and samples from industries that use H2O2 as a bleach and germicide. Workers with little experience in sensor fabrication and limited funding will particularly benefit from utilization of the proposed H2O2 probes, which as well as being used in H2O2 testing also have a potential application as the transducer unit of oxidase-based biosensors with amperometric H2O2 readout.

An electrochemical method for detecting the biomarker 4-HPA by allosteric activation of Acinetobacterbaumannii reductase C1 subunit.

The C1 (reductase) subunit of 4-hydroxy-phenylacetate (4-HPA) 3-hydroxylase (HPAH) from the soil-based bacterium Acinetobacterbaumannii catalyzes NADH oxidation by molecular oxygen, with hydrogen peroxide as a by-product. 4-HPA is a potent allosteric modulator of C1, but also a known urinary biomarker for intestinal bacterial imbalance and for some cancers and brain defects. We thus envisioned that C1 could be used to facilitate 4-HPA detection. The proposed test protocol is simple and in situ and involves addition of NADH to C1 in solution, with or without 4-HPA, and direct acquisition of the H2O2 current with an immersed Prussian Blue-coated screen-printed electrode (PB-SPE) assembly. We confirmed that cathodic H2O2 amperometry at PB-SPEs is a reliable electrochemical assay for intrinsic and allosterically modulated redox enzyme activity. We further validated this approach for quantitative NADH electroanalysis and used it to evaluate the activation of NADH oxidation of C1 by 4-HPA and four other phenols. Using 4-HPA, the most potent effector, allosteric activation of C1 was related to effector concentration by a simple saturation function. The use of C1 for cathodic biosensor analysis of 4-HPA is the basis of the development of a simple and affordable clinical routine for assaying 4-HPA in the urine of patients with a related disease risk. Extension of this principle to work with other allosteric redox enzymes and their effectors is feasible.

Amperometric enzymatic sensing of glucose using porous carbon nanotube films soaked with glucose oxidase.

Glucose oxidase was soaked into a porous carbon nanotube film coating on a platinum disk electrode, then trapped beneath a topcoat of electrodeposition paint. The resulting sensors, operated at a potential of +0.6 V (vs. Ag/AgCl), produced a glucose signal that was linear up to 40 mM, with a 50 µM detection limit. Signal stability over >100 h of continuous operation in a flow cell showed the remarkable functional durability of the biosensor, and confirmed that the electropaint coating effectively prevented loss of the enzyme. This performance is deemed to derive from the minimalistic immobilization layer design and the prevention of protein leakage. The immobilization method has a potentially wide scope, in that it may also be applicable in other types of enzymatic biosensor. Graphical abstract Illustration of an enzyme biosensor design that uses glucose oxidase in bare carbon nanotube electrode modifications with electropaint topcoat for amperometric glucose quantification. Immobilization matrix supplementation with extra functional (nano-) materials was unnecessary for high-quality and stable analysis performance.

Amperometric Detection of the Urinary Disease Biomarker p-HPA by Allosteric Modulation of a Redox Polymer-Embedded Bacterial Reductase.

We report an amperometric biosensor for the urinary disease biomarker para-hydroxyphenylacetate ( p-HPA) in which the allosteric reductase component of a bacterial hydroxylase, C1-hpah, is electrically wired to glassy carbon electrodes through incorporation into a low-potential Os-complex modified redox polymer. The proposed biosensing strategy depends on allosteric modulation of C1-hpah by the binding of the enzyme activator and analyte p-HPA, stimulating oxidation of the cofactor NADH. The pronounced concentration-dependence of allosteric C1-hpah modulation in the presence of a constant concentration of NADH allowed sensitive quantification of the target, p-HPA. The specific design of the immobilizing redox polymer with suitably low working potential allowed biosensor operation without the risk of co-oxidation of potentially interfering substances, such as uric acid or ascorbic acid. Optimized sensors were successfully applied for p-HPA determination in artificial urine, with good recovery rates and reproducibility and sub-micromolar detection limits. The proposed application of the allosteric enzyme C1-hpah for p-HPA trace electroanalysis is the first successful example of simple amperometric redox enzyme/redox polymer biosensing in which the analyte acts as an effector, modulating the activity of an immobilized biocatalyst. A general advantage of the concept of allosterically modulated biosensing is its ability to broaden the range of approachable analytes, through the move from substrate to effector detection.

Tuned Amperometric Detection of Reduced ß-Nicotinamide Adenine Dinucleotide by Allosteric Modulation of the Reductase Component of the p-Hydroxyphenylacetate Hydroxylase Immobilized within a Redox Polymer.

We report the fabrication of an amperometric NADH biosensor system that employs an allosterically modulated bacterial reductase in an adapted osmium(III)-complex-modified redox polymer film for analyte quantification. Chains of complexed Os(III) centers along matrix polymer strings make electrical connection between the immobilized redox protein and a graphite electrode disc, transducing enzymatic oxidation of NADH into a biosensor current. Sustainable anodic signaling required (1) a redox polymer with a formal potential that matched the redox switch of the embedded reductase and avoided interfering redox interactions and (2) formation of a cross-linked enzyme/polymer film for stable biocatalyst entrapment. The activity of the chosen reductase is enhanced upon binding of an effector, i.e. p-hydroxy-phenylacetic acid ( p-HPA), allowing the acceleration of the substrate conversion rate on the sensor surface by in situ addition or preincubation with p-HPA. Acceleration of NADH oxidation amplified the response of the biosensor, with a 1.5-fold increase in the sensitivity of analyte detection, compared to operation without the allosteric modulator. Repetitive quantitative testing of solutions of known NADH concentration verified the performance in terms of reliability and analyte recovery. We herewith established the use of allosteric enzyme modulation and redox polymer-based enzyme electrode wiring for substrate biosensing, a concept that may be applicable to other allosteric enzymes.

Automated Quantitative Enzyme Biosensing in 24-Well Microplates.

We report a novel system for glucose estimation in model and real samples, utilizing enzyme-modified pencil leads (PL) as effective electrochemical biosensors for robotic substrate quantification in 24-well microplates. Electrochemically formed carboxyl groups on the surface of the graphite were cross-linked to amino groups in the enzyme so as to attach glucose oxidase to the PL surface. Automated amperometric sensing of glucose solutions in microtiter-plate wells used computer-controlled stepper motors to move the biosensor/counter/reference electrode assemblies sequentially between the samples. This setup achieved stable analyte response and, in calibration trials, a linear response range and detection limit of 0.1-8 mM and 0.05 ± 0.01 mM, respectively. The biosensor microplate assay offered accurate "hands-off" evaluation of 4 or 20 samples per plate run, in the standard addition or calibration curve mode, respectively. Mode-independent glucose assays in standard solutions and human serum samples worked reproducibly with close to 100% recovery. The choice of cheap and practical PL enzyme biosensors and simple nonmicrofluidic measurement automation offers a convenient, labor- and cost-efficient form of quantitative biosensing, with a reduced risk of operator errors. The robotic approach is best suited to repetitive measurements of sample series, with academic research and clinical, environmental, pharmaceutical, or biotechnological analysis being potential areas for future exploitations of the methodology.