Microchannel conductivity measurements in microchip for on line monitoring of dephosphorylation rates of organic phosphates using paramagnetic-beads linked alkaline phosphatase

Abstract

This paper presents the use of polymer coated microelectrodes for the realtime conductivity monitoring in a microchannel photoablated through the polymer without contact. Based on this strategy, a small conductometry sensor has been developed to record in time conductivity variation when an enzymatic reaction occurs through the channel. The rate constant determination, k2, for the dephosphorylation of organic phosphate-alkaline phosphatase-superparamagnetic beads complex using chemically different substrates such as adenosine monoesterphosphate, adenosine diphosphate and adenosine triphosphate was taken as an example to demonstrate selectivity and sensivity of the detection scheme. The k2 value measured for each adenosine phosphate decreases from 39 to 30 s(-1) in proportion with the number (3, 2 and 1) of attached phosphate moiety, thus emphasizing the steric hindrance effect on kinetics.

Finite element modelling of non-faradic electric impedance spectroscopy through flexible polymer microchip

“…The value of the slope obtained from the fit of the experimental data corresponds to the microchannel cell constant and is equal to 8×10 4 m -1 . The modeling cell constant is slightly lower than the experimental one previously determined (5×10 3 m -1 ) [25]. The discrepancy between the experimental and modelled apparent cell-constant originates from the resistive contribution of the photoablated PET surface, which has been neglected in our simulations.…”

“…[22] This method could be used for real time monitoring of microchannel dielectric property changes i.e. its interfacial impedance in the low frequencies (LF) and resistance variations in the high frequencies (HF), while a chemical reaction was taking place at the functionalized PET surface (adsorption monitoring) [23], [24] or in the microchannel bulk (conductimetry), [15], [25], respectively. For instance, the interfacial capacitance changes during binding events (between two proteic ligands) were reported for the detection and quantification of a protein attached on PET by using its corresponding rabbit antiprotein antibodies as an analyte in the flow microchannel.…”

“…When an enzyme-substrate in the mobile phase (monoesterphosphate) comes in contact with the enzyme biocatalytic center, the dephosphorylation reaction starts and the inorganic phosphate ions produced lead to an increase of the conductivity. [22], [15], [25] In this application, the great advantage of the mutual use of contactless microelectrodes and specific ligand linked to microbeads is to prevent microelectrodes fouling. Indeed, it is difficult to regenerate microelectrode surfaces in small channels.…”

“…Here, the multiple use of the same PET microchip is possible, which also results in a lower risk of biological sample damage due to an enzyme activity lost. [25].…”

Quenching of graphene quantum dots fluorescence by alkaline phosphatase activity in the presence of hydroquinone diphosphate

“…Consequently, due to the relevance of ALP in clinical diagnosis, food safety and also in bioscience there is a growing interest in the development of techniques for ALP detection in a sensitive, fast and easy manner. The commonly reported monitoring techniques for ALP are based on the colorimetric, chemiluminescent, fluorometric, surface‐enhanced Raman spectroscopy or electrochemical detection of the products of ALP reaction with specific substrates. However, most of them, present several disadvantages since they are expensive, time‐consuming, or require bulky instrumentation.…”