Highly crosslinked and clickable poly(divinylbenzene) microspheres by type II photoinitiated precipitation polymerization

A new method for the synthesis of highly crosslinked and in situ alkyne functionalized microspheres, by combining type II photoinitiation with precipitation polymerization, is reported. Using a benzophenone-tertiary amine initiation system, the method allows the preparation of functional microspheres at room temperature by UV irradiation in the absence of stabilizers or surfactants of any kind. To demonstrate the presence and accessibility of the alkyne moiety on the polymer particles, fluorescent pyrene groups are attached to the microspheres by copper(I)-catalyzed alkyne-azide cycloaddition click reaction, using 1-azidomethyl pyrene as a model compound. FT-IR, fluorescence spectroscopy, fluorescence microscopy, and scanning electron microscopy are used for characterization of the particles and to monitor the click reaction

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

Molecularly imprinted polymers for small cell lung cancer diagnosis

According to the World Health Organisation, cancer is the second leading cause of death globally and lung cancer is the most common type of cancer. Despite the progress that has been made over the years, there is still a need for improved early-stage diagnostic tools and better cancer treatment options. The detection of cancer at an early stage is of particular importance for aggressive cancer types, such as Small Cell Lung Cancer (SCLC), since intervention with therapy at an early-stage is critical for patient survival. In this thesis, the design and synthesis of highly crosslinked, magnetic and non-magnetic molecularly imprinted polymer (MIP) microspheres for exploitation in the early-stage diagnosis of SCLC is described, where a bottom-up proteomics approach was taken to target the signature peptide of the SCLC biomarker neuron-specific enolase (NSE). A prime focus was on the synthesis of MIPs using a template analogue approach, but also the production of microspheres with diameters around 2 μm since such particles are well-suited to demanding chemical separation work. MIPs and their non-imprinted analogues were synthesised by precipitation polymerisation using free radical polymerisation, photoinitiated polymerisation, and atom transfer radical polymerisation (ATRP), and packed into columns for on-line molecularly imprinted solid-phase extraction (MISPE) LC-MS/MS. The polymers had selectivity for the NSE signature peptide target, which enabled it to be extracted efficiently from water-rich samples, allowing its determination at clinically relevant levels. Next, the first example of activators regenerated by electron transfer atom transfer (ARGET) ATRP under precipitation polymerisation (PP) conditions is reported. This new method for polymer synthesis (called ARGET ATRPP) requires only very low levels of metal catalyst for success, a distinctive feature that enables the production of uniform polymer microspheres under reversible-deactivation radical polymerisation control but without a requirement to remove dissolved molecular oxygen rigorously from monomer solutions before polymerisation.According to the World Health Organisation, cancer is the second leading cause of death globally and lung cancer is the most common type of cancer. Despite the progress that has been made over the years, there is still a need for improved early-stage diagnostic tools and better cancer treatment options. The detection of cancer at an early stage is of particular importance for aggressive cancer types, such as Small Cell Lung Cancer (SCLC), since intervention with therapy at an early-stage is critical for patient survival. In this thesis, the design and synthesis of highly crosslinked, magnetic and non-magnetic molecularly imprinted polymer (MIP) microspheres for exploitation in the early-stage diagnosis of SCLC is described, where a bottom-up proteomics approach was taken to target the signature peptide of the SCLC biomarker neuron-specific enolase (NSE). A prime focus was on the synthesis of MIPs using a template analogue approach, but also the production of microspheres with diameters around 2 μm since such particles are well-suited to demanding chemical separation work. MIPs and their non-imprinted analogues were synthesised by precipitation polymerisation using free radical polymerisation, photoinitiated polymerisation, and atom transfer radical polymerisation (ATRP), and packed into columns for on-line molecularly imprinted solid-phase extraction (MISPE) LC-MS/MS. The polymers had selectivity for the NSE signature peptide target, which enabled it to be extracted efficiently from water-rich samples, allowing its determination at clinically relevant levels. Next, the first example of activators regenerated by electron transfer atom transfer (ARGET) ATRP under precipitation polymerisation (PP) conditions is reported. This new method for polymer synthesis (called ARGET ATRPP) requires only very low levels of metal catalyst for success, a distinctive feature that enables the production of uniform polymer microspheres under reversible-deactivation radical polymerisation control but without a requirement to remove dissolved molecular oxygen rigorously from monomer solutions before polymerisation

Synthesis of uniform polymer microspheres with "living" character using ppm levels of copper catalyst : ARGET atom transfer radical precipitation polymerisation

The first example of activators regenerated by electron transfer atom transfer radical polymerisation (ARGET ATRP) under precipitation polymerisation (PP) conditions is reported. This new method for polymer synthesis (called ARGET ATRPP) requires only very low levels of metal catalyst for success, a distinctive feature that enables the production of uniform polymer microspheres under reversible-deactivation radical polymerisation control. The influence of the polymerisation conditions (monomer concentration, reaction time, initiator and catalyst concentrations) on the polymerisations and microsphere products were investigated and optimised. The ARGET ATRPP methodology was used to prepare polymer microspheres with narrow particle size distributions and mean particle diameters in the micron-size range, under dilute monomer conditions (2%) whilst using very low copper catalyst concentrations (down to 1.7 ppm). The "living" characteristics of the polymer microspheres enabled poly(methyl methacrylate) brushes to be grafted-from the microspheres directly without the need for any additional surface functionalisation step to immobilise initiator moieties

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

Poly(o-aminophenol) prepared by Cu(II) catalyzed air oxidation and its use as a bio-sensing architecture

A novel procedure for the preparation of organosoluble polyo-aminophenol) is described. The method pertains to the reduction of CuBr2 with o-amino phenol OAP) at room temperature. The air oxidation of the CuI) species to Cu(II) facilitated the formation of polyo-aminophenol) POAP) in repeated cycles. The obtained polymer, soluble in common organic solvents such as dimethylformamide, acetone, and chloroform, was characterized by spectral analyses. It was found that both quinoid and benzoid structures were present in the polymer chain. Polyo-aminophenol) architecture was then applied as an immobilization matrix to obtain a long-lived and fast response bio-sensing system. Glucose oxidase GOx) was utilized as a model enzyme for the detection of glucose in beverages. After the optimization studies, the surface characterization of POAP/GOx was performed by scanning electron microscopy SEM) measurements. Under the optimized conditions, the biosensor exhibited a rapid response 2 s), a low detection limit 0.02 mM), and high sensitivity 79.80 mu A mM(-1) cm(-2)). The K-M(app) value was determined as 0.16 mM. Furthermore, there was no considerable activity loss for 15 days for the POAP/GOx biofilm indicating excellent recovery as tested with real samples

Simultaneous and Sequential Synthesis of Polyaniline-g-poly(ethylene glycol) by Combination of Oxidative Polymerization and CuAAC Click Chemistry: A Water-Soluble Instant Response Glucose Biosensor Material

A novel approach for the in situ synthesis of conjugated polyaniline-poly(ethylene glycol) graft copolymer (PA-g-PEG) by the combination of oxidative polymerization and copper catalyzed azide alkyne cycloaddition (CuAAC) click reaction is described. The method pertains to the reduction of the CuBr2 catalyst during the oxidative copolymerization of aniline and aminophenyl propargylether to Cu(I) species, which catalyze the CuAAC reaction between thus formed polyaniline with pendant alkyne groups and independently prepared azide functional PEG in both simultaneous and sequential manner. The obtained water-soluble (PA-g-PEG) was used for the construction of glucose biosensor by a simple one-step approach. Combined electrostatic polyanion-polycation and hydrogen bond interactions between (PA-g-PEG) and glucose oxidase provided a suitable immobilization matrix for the enzyme resulting in excellent analytical parameters. (PA-g-PEG) based glucose biosensor exhibited a remarkable response time, producing an instant signal upon addition of analyte, making this sensor an attractive alternative for the existing devices

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