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The reliable identification and quantification of phosphorylated amino acids, peptides and proteins is one of the key challenges in contemporary bioanalytical research, noteworthy, to diagnose and treat diseases at an early developmental stage. Miniaturised sensing devices like microfluidic chips combined with “smart” detection chemistry, simple data assessment, processing and presentation are very attractive for benchtop use in clinical environments.
We developed novel synthetic probes targeting phosphorylated amino acids, based on core-shell microparticles consisting of a silica core coated with a molecularly imprinted polymer (MIP) shell. These “plastic antibodies” are extremely robust, resist denaturing solvents and elevated temperatures, can be reproducibly produced at low cost, and potentially overcome many of the practical problems in current bioanalytical detection strategies. The MIP layer contains a fluorescent probe monomer, binds selectively to phosphorylated tyrosine (pTyr) with a significant imprinting factor higher than 3.5 and responds with a “lighting-up” of its fluorescence accompanied by the development of a strongly red-shifted emission band toward the analyte.
In analogy to our previous work [4], the bead-based ratiometric detection scheme has also been successfully transferred to a microfluidic chip format to demonstrate its applicability to rapid assays. Such a miniaturised device could yield an automated pTyr measurement system in the future. The setup was built by coupling a modular microfluidic system [5] for amino acid functionalisation (Fmoc protection) and a multi-layer PDMS/Teflon/glass microfluidic chip [6] for buffering, extraction (micropillars co-flow extraction) and selective adsorption on the MIP core-shell particles.
A miniaturised optical assembly for low-light fluorescence measurements was also developed. Based on small opto-electronic parts and optical fibres, the emission from the MIP particles upon addition of pTyr concentrations from 0.5 – 200 μM could be monitored in real-time.
The reliable identification and quantification of phosphorylated amino acids, peptides and proteins is one of the key challenges in contemporary bioanalytical research, noteworthy, to diagnose and treat diseases at an early developmental stage1. Miniaturised sensing devices like microfluidic chips combined with “smart” detection chemistry, simple data assessment, processing and presentation are very attractive for benchtop use in clinical environments.
We developed novel synthetic probes targeting phosphorylated amino acids, based on core-shell microparticles consisting of a silica core coated with a molecularly imprinted polymer (MIP) shell. These “plastic antibodies” are extremely robust, resist denaturing solvents and elevated temperatures, can be reproducibly produced at low cost, and potentially overcome many of the practical problems in current bioanalytical detection strategies. The MIP layer contains a fluorescent probe monomer, binds selectively to phosphorylated tyrosine (pY) with a significant imprinting factor higher than 3.5 and responds with a “lighting-up” of its fluorescence accompanied by the development of a strongly red-shifted emission band toward the analyte. In analogy to our previous work4, the bead-based ratiometric detection scheme has also been successfully transferred to a microfluidic chip format to demonstrate its applicability to rapid assays. Such a miniaturised device could yield an automated pY measurement system in the future. The setup was built by coupling a modular microfluidic system5 for amino acid functionalisation (Fmoc protection) and, as shown in Figure 1, a multi-layer PDMS/Teflon/glass microfluidic chip6 for buffering, extraction (micropillars co-flow extraction) and selective adsorption on the MIP core-shell particles.
A miniaturised optical assembly for low-light fluorescence measurements was also developed. Based on small opto-electronic parts and optical fibres, the emission from the MIP particles upon addition of pY concentrations from 0.5-200 μM could be monitored in real-time.
Cancer is a leading cause of death worldwide, and its early detection and resultant treatment contributes significantly to patient recovery and survival. Detection is currently based on magnetic resonance imaging and computed tomography, methods that are expensive, while processing of the results is time-consuming1. There is a need for low-cost cancer detection techniques that give conclusive results in the shortest time possible. When equipped with a reporter function, molecularly imprinted polymers (MIPs) targeting tumor markers on cancerous cells may provide a cheaper solution for imaging-based cancer detection. Thin MIP layers immobilized on particle platforms are ideal in this regard, because a fluorescence reporter can be integrated into the particle core and/or MIP shell and such core/shell nanoparticles show faster response times and increased selectivity in comparison to bulk MIPs.
Changes in sialylation patterns of cell surface glycans indicate malignancy2. Here, we present the development of MIPs that target sialic acid-terminated glycans (SA MIPs), prepared as a thin layer on a polystyrene core/silica shell nanoparticle platform. The MIP particles contain fluorescent emitters and can be applied in fluorescence imaging of malignant tumors. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) are used for structural characterization. Binding capacity of the MIPs to target glycans and competing sugars is also evaluated and compared to that of the corresponding non-imprinted polymer particles (NIP).
Digital holographic cytometry (DHC) is a state-of-the-art quantitative Phase imaging (QPI) method that permits time-lapse imaging of cells without induced cellular toxicity. DHC platforms equipped with semi-automated image segmentation and analysis software packages for assessing cell behavior are commercially available. In this study we investigate the possible uptake of nanoprobes in macrophages in vitro over time.