Use of voltammetric solid-state (micro)electrodes for studying biogeochemical processes: Laboratory measurements to real time measurements with an in situ electrochemical analyzer (ISEA)

Solid-state voltammetric (micro)electrodes have been used in a variety of environments to study biogeochemical processes. Here we show the wealth of information that has been obtained in the study of sediments, microbial mats, cultures and the water column including hydrothermal vents. Voltammetric analyzers have been developed to function with operator guidance and in unattended mode for temporal studies with an in situ electrochemical analyzer (ISEA). The electrodes can detect the presence (or absence) of a host of redox species and trace metals simultaneously. The multi-species capacity of the voltammetric electrode can be used to examine complex heterogeneous environments such as the root zone of salt marsh sediments. The data obtained with these systems clearly show that O2 and Mn2+ profiles in marine sedimentary porewaters and in microbial biofilms on metal surfaces rarely overlap indicating that O2 is not a direct oxidant for Mn2+. This lack of overlap was suggested originally by Joris Gieskes\u27 group. In waters emanating from hydrothermal vents, Fe2+, H2S and soluble molecular FeS clusters (FeSaq) are detected indicating that the reactants for the pyrite formation reaction are H2S and soluble molecular FeS clusters. Using the ISEA with electrodes at fixed positions, data collected continuously over three days near a Riftia pachyptila tubeworm field generally show that O2 and H2S anti-correlate and that H2S and temperature generally correlate. Unlike sedimentary environments, the data clearly show that Riftia live in areas where both O2 and H2S co-exist so that its endosymbiont bacteria can perform chemosynthesis. However, physical mixing of diffuse flow vent waters with oceanic bottom waters above or to the side of the tubeworm field can dampen these correlations or even reverse them. Voltammetry is a powerful technique because it provides chemical speciation data (e.g.; oxidation state and different elemental compounds/ions) as well as quantitative data. Because (micro)organisms occupy environmental niches due to the system\u27s chemistry, it is necessary to know chemical speciation. Voltammetric methods allow us to study how chemistry drives biology and how biology can affect chemistry for its own benefit

Advantages of Electro-deposited Gold on Carbon Electrodes for NT-proBNP Immunosensor for Development of Heart Failure Test Kit

Accurate measurement of the N-terminal pro B-type natriuretic peptide (NT-proBNP) in serum is important for the diagnosis of heart failure (HF). Carbon screen-printed electrodes (SPCEs) modified with graphene oxide (GO) or gold (Au) were compared for the construction of NT-proBNP immunosensors. NT-proBNP and its recognition unit, a single-chain variable fragment fused with alkaline phosphatase (scFv-AP), were expressed and purified. The currents of the electrodes immobilized with scFv-AP were measured after adding an ethanolamine (ETA), blank and NT-proBNP in either phosphate buffer saline (PBS) or human serum. SPCE/Au had lower mean baseline slopes than for SPCE/GO for all measurements, in both PBS and serum, indicating greater accuracy for SPCE/Au. None of the measurements in PBS had statistically different peak currents between SPCE/GO and SPCE/Au; however, there was a significant difference with the serum. The significant reduction of SPCE/GO peak currents after applying serum blank implied non-specific absorption on the surface. The peak current of 300 pg/mL of NT-proBNP in the serum measured on SPCE/Au was significantly higher (by a factor of three) than on SPCE/GO, suggesting the possibility of using SPCE/Au to detect NT-proBNP at higher concentrations. The binding efficiency of scFv-AP to NT-proBNP did not depend on the electrodes, as shown by the similar delta peak-currents (Blank-Target). Thus, immobilized scFv-AP on SPCE/Au electrodes had good potential to accurately detect NT-proBNP in serum, for use in the fabrication of an HF test kit

Generation of a Single-Chain Variable Fragment Antibody against Feline Immunoglobulin G for Biosensor Applications

For many decades, feline infectious disease has been among the most common health problems and a leading cause of death in cats. These diseases include toxoplasmosis, feline leukemia virus (FeLV), and particularly feline immunodeficiency virus (FIV) disease. Early diagnosis is essential to increase the chance of successful treatment. Generally, measurement of the IgG level is considered to be indicative of an individual’s immune status for a particular pathogen. The antibodies specific to feline IgG are crucial components for the development of a detection kit. In this study, feline IgG-bound scFv was selected using phage display technology. Three rounds of biopanning were conducted against purified feline IgG. Through an indirect enzyme-linked immunosorbent assay (ELISA), two scFv clones demonstrating the best binding ability to feline IgG were chosen for biochemical characterization. In addition, the selected scFv (N14) was expressed and purified in a bacterial system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the size of the purified N14 was 29 kDa. A sandwich ELISA was used to evaluate the binding capacity of the purified scFv to feline IgG. As expected, the purified N14 had the capacity to bind feline IgG. Furthermore, N14 was modified to create a scFv-alkaline phosphatase (scFv-AP) fusion platform. The surface plasmon resonance (SPR) results revealed that N14-AP bound to feline IgG with an affinity binding value of 0.3 ± 0.496 μM. Additionally, the direct ELISA demonstrated the binding capacity of N14-AP to feline IgG in both cell lysate and purified protein. Moreover, N14-AP could be applied to detect feline IgG based on electrosensing with a detection limit of 10.42 nM. Overall, this study successfully selected a feline IgG-bound scFv and developed a scFv-AP platform that could be further engineered and applied in a feline infectious disease detection kit