Novel and highly stable strategy for the development of microfluidic enzymatic assays based on the immobilization of horseradish peroxidase (HRP) into cotton threads.

The use of biological components in the development of new methods of analysis and point-of-care (POC) devices is an ever-expanding theme in analytical chemistry research, due to the immense potential for early diagnosis of diseases and monitoring of biomarkers. In the present work, the evaluation of an electrochemical microfluidic device based on the immobilization of horseradish peroxidase (HRP) enzyme into chemically treated cotton threads is described. This bioreactor was used as a channel for the build of the microfluidic device, which has allowed to use of a non-modified screen-printed electrode (SPE) as an amperometric detector. Cotton threads were treated using citric acid, and the immobilization of HRP has been performed by EDC/NHS crosslinking, connecting amine groups of the enzymes to carboxylic acids in the cellulosic structure. For the analytical evaluation, an amperometric assay for hydrogen peroxide detection was performed after the injection of H2O2 and hydroquinone (HQN) concomitantly. The enzymatic reaction consumes H2O2 leading to the formation of O-quinone, which is readily reducible at non-modified SPE. Several experimental parameters related to enzyme immobilization have been investigated and under the best set of conditions, a good analytical performance was obtained. In addition, the threads were freezer-stored and, after 12 weeks, 84% of hydrogen peroxide sensitivity was maintained, which is very reasonable for enzyme-based systems and still offers good analytical precision. Therefore, a simple and inexpensive microfluidic system was reported by crosslinking carboxylic groups to amine-containing macromolecules, suggesting a new platform for many other protein-based assays.

Microfluidic thread based electroanalytical system for green chromatographic separations.

The use of miniaturized chromatographic systems is an important strategy for reducing the consumption of supplies related to separations, allowing the development of more sustainable analytical methodologies. However, the high cost and complexity in the production of these systems combined with the operational difficulties and the need for the use of solvent and sample pretreatment are challenges to be overcome in order to make the chromatographic methods greener. Here, we report the construction and development of a low cost microfluidic system for green and solvent-free chromatographic separations with electrochemical detection integrated into cotton threads without the use of any mechanical pumping to transport the solutions. The manufacture of the proposed system was performed by simple assembly of the components, with the separation of the species based on an ion exchange mechanism and detection using gold electrodes manufactured directly on the cotton threads. A linear range of 0.025-5.0 mM was obtained for the effective separation of ascorbic acid (AA) and dopamine (DA) with detection limits of 2.89 µM (for AA) and 4.41 µM (for DA). Each analysis was performed at a low cost (less than 0.01 dollars), and with a small volume of waste generated (107.1 µL). So, the proposed system was successfully employed to determine the levels of AA and DA present in the tears of healthy volunteers without sample pretreatment, indicating the good analytical performance of the system and the possibility of performing greener chromatographic separations.