# Gel-Phase Microextraction Using Microfluidic-Directed Ultrashort Peptide Assemblies for the Determination of Drugs in Oral Fluids

## TL;DR

A new microfluidic method uses self-assembled peptides to extract drugs from saliva, offering a non-invasive way to detect medications.

## Contribution

A novel microfluidic-based extraction method using tripeptide hydrogels for drug detection in oral fluids is introduced.

## Key findings

- Peptide microfibers formed in situ in a microchannel can entrap drugs like 5-FU and naproxen with specific molar ratios.
- The hydrogel sorbent successfully extracted drugs from buffer and simulated saliva with measurable efficiency.
- 19F NMR spectroscopy on microcoils enabled quantitative drug analysis from small fluid volumes.

## Abstract

This study introduces an innovative microfluidic-based approach for extracting drugs from oral fluids using self-assembled tripeptide hydrogels as sorbents. Peptide microfiber derived from the heterochiral tripeptide DLeu-LPhe-LPhe was formed in situ within the 14 mm-long microchannel of a two-inlet microfluidic device. The methodology enables the laminar flow-driven mixing of buffer solutions, inducing hydrogel formation at their interface. The resulting fiber exhibited a well-defined morphology and β-sheet structure, confirmed by Raman spectroscopy and Thioflavin T fluorescence. The peptide fibers co-assembled successfully with 5-fluorouracil (5-FU) and naproxen (39.8 ± 1.4 nmol of 5-FU and 27.4 ± 6.6 nmol of naproxen per 112 nmol of peptide used to prepare the fiber), resulting in a molar ratio drug/peptide ratio of approximately 1:3 and 1:4, respectively, demonstrating versatility in drug entrapment. The use of the gel fiber as a sorbent phase was first assessed in buffer, and subsequently, the optimized method was applied to saliva. Adsorption studies under stopped-flow conditions showed a significant drug adsorption capability from buffered solutions by the pre-formed hydrogel (32.8 ± 0.9% of 5-FU and 36.4 ± 3.3% of naproxen per fiber preformed with 112 nmol of peptide), demonstrating their suitability as sorbent material. The extension of the methodology to simulated saliva samples allowed extraction of 36% of 5-FU by the fiber, as determined by 19F NMR spectroscopy on microcoils, which enabled us to work with the small volume of fluid extracted from the microfluidic device and provided clean spectra and quantitative results. These findings highlight the potential of this tripeptide hydrogel as a sorbent material for therapeutic drug monitoring and toxicological analysis via a simple, non-invasive and rapid approach for drug detection in oral fluids.