Abstract
In this work, we introduce a system combining an acoustic trap for bead injection with attenuated total reflection (ATR) infrared (IR) spectroscopy. By mounting an acoustofluidic cell hosting an ultrasound source on top of a custom-built ATR fixture we were able to trap beads labeled with the enzyme alkaline phosphatase without requiring any mechanical retention elements. Sequential injection analysis was employed for reproducible sample handling and bead injection into the acoustic trap. To showcase potential applications of the presented setup for kinetic studies, we monitored the conversion of p-nitrophenylphosphate into p-nitrophenol and phosphate via beads carrying the immobilized enzyme using ATR-IR spectroscopy. Retaining the labeled beads via ultrasound particle manipulation resulted in excellent experimental reproducibility (relative standard deviation, 3.91%). It was demonstrated that trapped beads remained stably restrained with up to eight cell volumes of liquid passing through the acoustofluidic cell. Beads could be discarded in a straightforward manner by switching off the ultrasound, in contrast to systems containing mechanical retention elements, which require backflushing. Multiple experiments were performed by employing different substrate concentrations with the same batch of trapped beads as well as varying the amount of enzyme present in the cell, enabling enzyme kinetic studies and emphasizing the application of the proposed setup in studies where enzymatic reuse is desired. This proves the potential of the acoustic trap combined with ATR-IR spectroscopy to monitor the activity of immobilized enzymes and its ability to perform complex bead-based assays.
Highlights
In this work, we introduce a system combining an acoustic trap for bead injection with attenuated total reflection (ATR) infrared (IR) spectroscopy
We demonstrate the implementation of an acoustic trap for bead injection ATR-IR spectroscopy To showcase the applicability of the presented approach for monitoring enzymatic reactions, the substrate conversion induced by the trapped beads labeled with the enzyme alkaline phosphatase (AP) was monitored by ATR-IR spectroscopy
This work introduced the combination of an acoustic trap with ATR-IR spectroscopy, which is a promising alternative to established bead injection manifold approaches
Summary
We introduce a system combining an acoustic trap for bead injection with attenuated total reflection (ATR) infrared (IR) spectroscopy. We demonstrate the implementation of an acoustic trap for bead injection ATR-IR spectroscopy To showcase the applicability of the presented approach for monitoring enzymatic reactions, the substrate conversion induced by the trapped beads labeled with the enzyme alkaline phosphatase (AP) was monitored by ATR-IR spectroscopy. Suspension was injected into the acoustofluidic cell, where the beads were retained acoustically while their hosting liquid was discarded.
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