Fluorescent core-shell magnetic nanoparticles by type II photoinitiated polymerisation.

We report a fluorescent monomer-free method for the synthesis of fluorescent and stable magnetic nanocomposites using a benzophenone/rhodamine B bimolecular photoinitiator system. The method allows the synthesis of a fluorescent polymer shell layer around magnetic nanoparticles in one step by UV irradiation at ambient temperature.

One-Step Synthesis of Fluorescent Poly(divinylbenzene) Particles without Fluorescent Monomers.

A simple and cost-efficient method for fluorescent microsphere synthesis, which does not require any fluorescent monomers or modification steps to incorporate fluorescent moieties into the polymer particles, is reported. Using rhodamine B and benzophenone as bimolecular initiation system in type II photoinitiated precipitation polymerization, the method enables the preparation of fluorescent microspheres in one step, at room temperature and without the need for a stabilizer or surfactant of any type.

Photoinduced synthesis of fluorescent hydrogels without fluorescent monomers.

A fluorescent monomer-free one-step strategy is developed for the synthesis of fluorescent acrylamide gels, using inexpensive and commercially available rhodamine B as the hydrogen donor in a type II photoinitiation system. The obtained hydrogels are fluorescent and have limited fluorophore leaching over time, due to the covalent bond formed between the polymer network and rhodamine dye.

On-line duplex molecularly imprinted solid-phase extraction for analysis of low-abundant biomarkers in human serum by liquid chromatography-tandem mass spectrometry.

In the present work, a pair of molecularly imprinted polymers (MIPs) targeting distinct peptide targets were packed into trap columns and combined for automated duplex analysis of two low abundant small cell lung cancer biomarkers (neuron-specific enolase [NSE] and progastrin-releasing peptide [ProGRP]). Optimization of the on-line molecularly imprinted solid-phase extraction (MISPE) protocol ensured that the MIPs had the necessary affinity and selectivity towards their respective signature peptide targets - NLLGLIEAK (ProGRP) and ELPLYR (NSE) - in serum. Two duplex formats were evaluated: a physical mixture of the two MIPs (1:1 w/w ratio) inside a single trap column, and two separate MIP trap columns connected in series. Both duplex formats enabled the extraction of the peptides from serum. However, the trap columns in series gave superior extraction efficiency (85.8±3.8% and 49.1±6.7% for NLLGLIEAK and ELPLYR, respectively). The optimized protocol showed satisfactory intraday (RSD≤23.4 %) and interday (RSD≤14.6%) precision. Duplex analysis of NSE and ProGRP spiked into digested human serum was linear (R2≥0.98) over the disease range (0.3-30 nM). The estimated limit of detection (LOD) and limit of quantification (LOQ) were 0.11 nM and 0.37 nM, respectively, for NSE, and 0.06 nM and 0.2 nM, respectively, for ProGRP. Both biomarkers were determined at clinically relevant levels. To the best of our knowledge, the present work is the first report of an automated MIP duplex biomarker analysis. It represents a proof of concept for clinically viable duplex analysis of low abundant biomarkers present in human serum or other biofluids.

Facilitating serum determination of neuron specific enolase at clinically relevant levels by coupling on-line molecularly imprinted solid-phase extraction to LC-MS/MS.

The identification and quantification of biomarkers is essential for the diagnosis, treatment, and long-term monitoring of many human diseases. In the present work, macromolecular synthetic receptors with pre-determined affinity and selectivity for the signature peptide of a prognostically significant small cell lung cancer (SCLC) biomarker - neuron-specific enolase (NSE) - were prepared in a porous polymer microsphere format using a template-directed synthesis strategy performed under precipitation polymerization conditions. The polymer microspheres were packed into short trap columns and then exploited as molecularly selective sorbents in a fully automated, on-line molecularly imprinted solid-phase extraction (MISPE) protocol. The on-line MISPE protocol was optimised with respect to the composition of the loading mobile phase, the flow rate, and the extraction time. The molecularly imprinted polymers (MIPs) showed high affinity and useful selectivity for the peptide target - the hexapeptide ELPLYR - compared to non-imprinted control polymers. The MIPs were able to retain the biomarker on-column for extraction times of up to 20 min, and the on-line MISPE method enabled complete recovery of the biomarker over the linear range 10-100 ng mL-1 when the biomarker was present in spiked ammonium bicarbonate solution (R2 = 0.994). For extractions of ELPLYR from very complex biological matrices, the recoveries of ELPLYR from reversed-phase SPE (RP-SPE)-treated and untreated digested human serum were 100.8 ± 6.2% and 61.6 ± 1.9%, respectively. Extractions of ELPLYR from spiked untreated digested serum were linear in the range of 7.5-375 ng mL-1 (R2 = 0.99). The limit of detection (LOD) and limit of quantification (LOQ) for the biomarker in digested serum were estimated to be 1.8 ng mL-1 and 6.0 ng mL-1, respectively, which is below the median reference level of NSE in humans (8.6 ng mL-1). This work sets in place the basis for a new diagnostic tool for SCLC that is sensitive, robust, automated, and antibody-free, and which works very well with complex human plasma samples.