Automated Quantitative Enzyme Biosensing in 24-Well Microplates

Abstract

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.

Laser-Cut Polymer Tape Templates for Scalable Filtration Fabrication of User-Designed and Carbon-Nanomaterial-Based Electrochemical Sensors

“…The results are generally in accordance with the F−C method (Table S2). In summary, the combination of droplet cell arrays on addressable SWNT sensors with inexpensive addressing instruments, e.g., a 3D printer reported here or previously reported computercontrolled stepper motors 46 , enables the construction of high-throughput electrochemical sensing systems with a simple instrument, high detection speed, and low cost.…”

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

“…The relative strengths of five phenols as activators of C1-catalyzed NADH corresponded with those determined using optical assays, with 4-HPA the most potent. The proposed methodology is thus applicable to screening allosteric redox enzymes for practical applications, especially when combined with robotic electroanalysis (36)(37)(38)(39)(40)(41)(42) or electrochemical minicells for fluid testing (43)(44)(45). The results of this study may encourage consideration of voltammetric or amperometric detection as an economical and convenient alternative to spectroscopic methods, for initial screening of library members from engineered enzyme synthesis though not necessarily for detailed kinetic or mechanistic studies.…”

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

“…A thin adherent film of CNTs was then formed on the Pt disks through a simple drop-and-dry step with 5 μL of a freshly ultrasonicated and thus homogenous 5 mg mL –1 suspension of the nanomaterial in DI water. 36 Next, the nano-porous CNT electrode deposit was loaded with GO x in a second drop-and-dry step with 5 μL of a 20 mg mL –1 solution of the enzyme in 0.1 M Na-PB. A final drop-and-dry step with 5 μL of a 10,000× dilution of the viscous commercial PAA suspension (0.5 mg mL –1 ) formed the polymeric topcoat that protected against loss of GO x by leakage.…”

“…Data acquisition was controlled by the Palmsense-4 software PSTrac5.8 , and individual measurements detected the glucose response of the PAA/GO x -CNT/Pt biosensors as anodic hydrogen peroxide (H 2 O 2 ) oxidation currents through an adjustment of the WE potential to +0.6 V versus RE, this potential being chosen from experience gained in previous glucose biosensing studies. 36 − 40 …”