Combining a hybrid chip and tube microfluidic system with fluorescent molecularly imprinted polymer (MIP) core-shell particles for the derivatisation, extraction, and detection of peptides with N-terminating phosphorylated tyrosine.

Abstract

The reliable identification and quantitation of phosphorylated amino acids, peptides and proteins is one of the key challenges in contemporary bioanalytical research, an area of particular interest when attempting to diagnose and treat diseases at an early stage. We have developed a synthetic probe for targeting phosphorylated amino acids, based on core-shell submicron-sized particles consisting of a silica core, coated with a molecularly imprinted polymer (MIP) shell. The MIP layer contains a fluorescent probe crosslinker which binds selectively to phosphorylated tyrosine (<b>pY</b>) moieties with a significant imprinting factor (IF) and responds with a "light-up" fluorescence signal. The bead-based ratiometric detection scheme has been successfully transferred to a microfluidic chip format and its applicability to rapid assays has been exemplarily shown by discriminating a <b>pY</b>-terminating oligopeptide against its non-phosphorylated counterpart. Such miniaturised devices could lead to an automated <b>pY</b> or <b>pY</b> N-terminated peptide measurement system in the future. The setup combines a modular microfluidic system for amino acid derivatisation, extraction (by micropillar co-flow) and selective adsorption and detection with the fluorescent MIP core-shell particle probes. A miniaturised optical assembly for low-light fluorescence measurements was also developed, based on miniaturised opto-electronic parts and optical fibres. The emission from the MIP particles upon binding of <b>pY</b> or <b>pY</b> N-terminated peptides could be monitored in real-time.