Electrochemical assay of sorbitol dehydrogenase at PEDOT modified electrodes - a new milk biomarker for confirmation of pregnancy in dairy cattle.

A robust electrochemical assay for sorbitol dehydrogenase (SORDH) activity in milk was developed using voltammetry and chronocoulometry at bare and polymer modified transducers. The motivation for the work was to evaluate the potential of SORDH as an early biomarker of bovine pregnancy using milk as sample matrix. SORDH is an enzyme involved in carbohydrate metabolism converting sorbitol, the sugar alcohol form of glucose, into fructose, with NAD+ as a cofactor being simultaneously reduced to NADH. The assay was optimised via direct NADH oxidation on glassy carbon and screen printed carbon electrodes followed by electropolymerisation of 3,4-ethylenedioxythiophene (EDOT) monomer to form an NADH responsive PEDOT surface which operated well in undiluted milk samples. Assay conditions such as incubation time and temperature were optimised resulting in a 3 min assay at 37 °C in the presence of 10 mM NAD+ and 20 mM sorbitol co-substrates, enabling NADH electro oxidation (linear range 0.25-5 mM, sensitivity 9.17 µC cm-2 mM-1 in undiluted milk). SORDH determination followed over the range 0.31-10 U mL-1 in milk samples with sensitivity 5.45 µC cm-2 U-1 mL with LOD 0.0787 U mL-1. The assay was applied to milk sample testing acquired as part of an approved animal study involving control and breeding cycles of dairy cows with focus on analysis at day 19 post artificial insemination. Significant differences between control and pregnant SORDH levels in whole milk animal samples were found (average values 2.57 and 4.07 ng mL-1 respectively), as verified using a commercial SORDH ELISA optical assay. Finally, progesterone monitoring over days 16-21 of the oestrous cycle employed an optical ELISA assay and confirmed maintenance of progesterone levels from day 19 onwards.

Impedimetric and electrochemical evaluation of a new redox active steroid derivative for hormone immunosensing.

Preparation and electrochemical interrogation of a novel redox active progesterone derivative progesterone thiosemicarbazone (PATC) is presented here together with an investigation into its suitability as conjugate in progesterone hormone immunosensing. PATC synthesis involved a condensation reaction between progesterone acetate and thiosemicarbazone hydrochloride. Voltammetric and pulse techniques confirmed the redox behaviour of the new compound with concentration and scan rate dependant irreversible behaviour evident at glassy carbon and gold transducers - ko (standard heterogeneous rate constant) was 2.56 × 10-3 cm2/s (ν = 100 mV/s in non-aqeuous media). Bioaffinity studies towards anti-progesterone antibodies involved a competitive ELISA format (optical) which confirmed recognition of the new progesterone derivative. Electrochemical impedance spectroscopy was employed as an interrogation technique in order to establish optimum binding and surface conditions for progesterone antigen-antibody interaction with the assistance of a redox probe (potassium hexacyanoferrate).