The goal of this research proposal is to advance our proprietary Lab-in-a-tube (Liat TM) system to include an embedded protein microarray for automated cardiac marker monitoring. The Liat system will allow us to perform various assays using whole blood in a """"""""sample-to-answer"""""""" format, while colloid protein coated arrays will allow for the simultaneous detection and quantification of various cardiac markers. The colloidal particles can adsorb one to two orders of magnitude more capture molecules than other protein coating methods and protein activity on the colloids is stable for over six months, even when stored under desiccated conditions. Moreover, the micropile structure created by the colloid particles may trap more analytes within a spot, resulting in an increase in the apparent affinity of the capture molecules to the analytes. Multiple fluorescence detection channels can be used to independently quantify possible interference factors and analytes, providing comprehensive quality control. Embedding these protein microarrays into Liat probes allows us to separate plasma from whole blood, perform binding assays under flow-conditions and detect binding events. We hypothesize that the use of lab-in-a-tube technology to create flow channels for performing the binding assays will eliminate the diffusion-rate limitation of array based assays and allow us to approximate the reaction-rate limited potential of bead based assays. The use of time resolved fluorescence labeling technology will further increase the sensitivity. To prove the concept, we will develop an array for testing brain natriuretic peptide, cardiac troponin I, and cardiac troponin T. Our Liat system has been demonstrated on rapid nucleic acid and cellular tests. This proposed research will further incorporate multiplex immunoassay functionality in the Liat system, and contribute to development of a uniform Liat Analyzer capable of nucleic acid, cellular, and protein tests.
