Lyophilization of chemiluminescent substrate reagents for high-sensitive microchannel-based lateral flow assay (MLFA) in point-of-care (POC) diagnostic system

Over the last few years, lateral flow assay (LFA) devices have grown to be the most common point-of-care test (POCT) platform facilitating disease diagnostics in low-resource environments. However, the lack of consistency and the limited sensitivity of these devices often lead to misdiagnosis and generates the need for an alternate approach. A chemiluminescence based microchannel-based lateral flow assay (MLFA) in a POCT platform can result in a much higher sensitivity but involves multiple additional steps of liquid reagents for the sequential execution of the signal amplification protocol. One of the best ways to develop a sample-to-answer system with minimum user intervention is to dry reagents on a chip prior to sample addition and to control the flow of the biological fluid through the drying chambers resulting in the reconstitution of the reagents. This work reports the methods for the successful lyophilization of the chemiluminescent substrate and its reconstitution in artificial serum without any significant loss of functionality. The lyophilized reagents were reconstituted and incorporated into the reaction chambers of a designed polymer lab-on-a-chip to implement a sandwich assay for the detection of malarial biomarkers. The results report a limit of detection (LOD) of 5.75 ng mL-1 which is sensitive enough to detect active malarial infection. Successful lyophilization and reconstitution of the chemiluminescent substrate, as reported here, can pave the way towards developing an autonomous POCT system implementing chemiluminescence based sandwich ELISA for enhanced sensitivity, portability, and ease-of-use in resource limited settings.