Antonio Guerreiro

Direct potentiometric quantification of histamine using solid-phase imprinted nanoparticles as recognition elements.

A new potentiometric sensor based on molecularly imprinted nanoparticles produced via the solid-phase imprinting method was developed. For histamine quantification, the nanoparticles were incorporated within a membrane, which was then used to fabricate an ion-selective electrode. The use of nanoparticles with high affinity and specificity allowed for label-free detection/quantification of histamine in real samples with short response times. The sensor could selectively quantify histamine in presence of other biogenic amines in real wine and fish matrices. The limit of detection achieved was 1.12×10(-6)molL(-1), with a linear range between 10(-6) and 10(-2)molL(-1) and a response time below 20s, making the sensor as developed a promising tool for direct quantification of histamine in the food industry.

Automatic reactor for solid-phase synthesis of molecularly imprinted polymeric nanoparticles (MIP NPs) in water

We report the development of an automated chemical reactor for solid-phase synthesis of MIP NPs in water. Operational parameters are under computer control, requiring minimal operator intervention. In this study, ready for use MIP NPs with sub-nanomolar affinity are prepared against pepsin A, trypsin and α-amylase in only 4 hours.

Influence of Surface‐Imprinted Nanoparticles on Trypsin Activity

Here, the modulation of enzyme activity is presented by protein-imprinted nanoparticles produced using a solid-phase approach. Using trypsin as target, binding of the nanoparticles to the enzyme results in its inhibition or in stabilization, depending on the orientation of the immobilized enzyme used during imprinting.

Introducing MINA – The Molecularly Imprinted Nanoparticle Assay

A new ELISA- (enzyme-linked immunosorbent assay)-like assay is demonstrated in which no elements of biological origin are used for molecular recognition or signaling. Composite imprinted nanoparticles that contain a catalytic core and which are synthesized by using a solid-phase approach can simultaneously act as recognition/signaling elements, and be used with minimal modifications to standard assay protocols. This assay provides a new route towards replacement of unstable biomolecules in immunoassays.

New potentiometric sensor based on molecularly imprinted nanoparticles for cocaine detection

Here we present a potentiometric sensor for cocaine detection based on molecularly imprinted polymer nanoparticles (nanoMIPs) produced by the solid-phase imprinting method. The composition of polymers with high affinity for cocaine was optimised using molecular modelling. Four compositions were selected and polymers prepared using two protocols: chemical polymerisation in water and UV-initiated polymerisation in organic solvent. All synthesised nanoparticles had very good affinity to cocaine with dissociation constants between 0.6nM and 5.3nM. Imprinted polymers produced in organic solvent using acrylamide as a functional monomer demonstrated the highest yield and affinity, and so were selected for further sensor development. For this, nanoparticles were incorporated within a PVC matrix which was then used to prepare an ion-selective membrane integrated with a potentiometric transducer. It was demonstrated that the sensor was able to quantify cocaine in blood serum samples in the range of concentrations between 1nM and 1mM.

Optimisation of the synthesis of vancomycin-selective molecularly imprinted polymer nanoparticles using automatic photoreactor

A novel optimized protocol for solid-state synthesis of molecularly imprinted polymer nanoparticles (nanoMIPs) with specificity for antibiotic vancomycin is described. The experimental objective was optimization of the synthesis parameters (factors) affecting the yield of obtained nanoparticles which have been synthesized using the first prototype of an automated solid-phase synthesizer. Applications of experimental design (or design of experiments) in optimization of nanoMIP yield were carried out using MODDE 9.0 software. The factors chosen in the model were the amount of functional monomers in the polymerization mixture, irradiation time, temperature during polymerization, and elution temperature. In general, it could be concluded that the irradiation time is the most important and the temperature was the least important factor which influences the yield of nanoparticles. Overall, the response surface methodology proved to be an effective tool in reducing time required for optimization of complex experimental conditions.

Molecularly imprinted polymers as a tool for the study of the 4-ethylphenol metabolic pathway in red wines.

A molecularly imprinted polymer (MIP) based methodology is described here for the determination of compounds that belong to the 4-ethylphenol (4EP) metabolic pathway in red wines. To this end, two MIP materials have been developed: a 4EP MIP as a class-selective material to extract phenols that belong to the 4EP metabolic pathway and a coumaric acid (CA) imprinted polymer as a MIP-based stationary phase capable of selectively separating these phenols on HPLC analysis, obtaining clean chromatograms. 4-vinyl pyridine and ethylene glycol dimethacrylate were respectively used as functional monomer and cross-linker for both MIPs. Once polymer compositions were optimised, the 4EP MIP was packed into SPE cartridges for wine sample clean-up and CA MIP was packed into HPLC columns to chromatographically separate the compounds present in the eluates obtained after SPE extraction. The accuracy of the proposed method was tested spiking wine samples with known concentrations of target compounds and subsequently, analytes were quantified by the standard addition method. Registered mean recoveries ranged from 95.2 to 109.2% and RSD values were below 10% in most cases. The described methodology was found to be suitable for the selective extraction and quantification of the compounds that belong to the 4EP metabolic pathway in red wines with minimal matrix effects and could be undoubtedly exploited to monitor 4EP and its precursors in wines.

Molecularly imprinted nanoparticles grafted to porous silica as chiral selectors in liquid chromatography

The work presented here explores the grafting of molecularly imprinted nanoparticles (MIN) on silica beads for the development of new chiral stationary phases (CSP). Both solid-phase imprinting and precipitation polymerisation were tested for MIN synthesis though the latter approach was the only one that provided efficient chiral selectors. MIN particles were prepared by iniferter polymerisation initiated by UV radiation, using itaconic acid as functional monomer and ethylene dimethacrylate as cross-linker. This resulted in particles with an average size of 249.0±4.0nm which were covalently immobilised onto chromatographic silica beads. The resultant CSP based on the composite silica beads-MIN was capable of resolving the racemate of the antidepressant drug citalopram and also separating its major metabolites by liquid chromatography, with better efficiency and peak symmetry than other MIP based CSP. The methodology presented here allowed for the quantification of the pharmacologically active enantiomer (+)-(S)-citalopram (SCIT) and its main metabolites (+)-(S)-desmethylcitalopram (SDCIT) and (+)-(S)-didesmethylcitalopram (SDDCIT) in urine, registering mean recoveries that ranged from 91.5 to 103.7% with RSD values that were below 10% in all tested concentration levels (0.1, 0.75 and 5μgmL-1), which confirmed method suitability for the intended application.

A pseudo-ELISA based on molecularly imprinted nanoparticles for detection of gentamicin in real samples

The enzyme-linked immunosorbent assay (ELISA) is one of the most widely employed tests in diagnostics, and it relies on the use of antibodies to quantify the molecule of interest. Molecularly imprinted nanoparticles (nanoMIPs), thanks to their stability, cost efficiency and easy production, are a promising alternative to antibodies in assays and sensors. In this work, nanoMIPs have been produced by means of a solid-phase approach and employed for the detection of gentamicin in real samples. The produced nanoMIPs were characterized using dynamic light scattering (DLS) and transmission electron microscopy (TEM) techniques. The determination of gentamicin in spiked milk was implemented through an assay similar to enzyme-linked immunosorbent assay, in which the nanoMIPs were used as a synthetic capture antibody (pseudo-ELISA). The detection of gentamicin was achieved in competitive binding experiments with a horseradish peroxidase–gentamicin conjugate. Gentamicin was determined in milk at clinically relevant concentrations with a mean accuracy of 94%. The cross-reactivity of such nanoparticles was investigated with streptomycin and ampicillin as control antibiotics, demonstrating excellent specificity.

Biocompatibility and internalization of molecularly imprinted nanoparticles

Molecularly imprinted polymers (MIP) are receiving increasing attention thanks to their robustness, stability, and inexpensive manufacture compared with their bio-analogues such as antibodies. The molecular imprinting process can be defined as the generation of molecular recognition sites in a synthetic polymer. The template-derived sites created within a polymeric matrix allow MIPs (often referred as plastic antibodies) to selectively recognize and bind to the target molecule. Therefore, MIPs can be used in sensors and in separation and diagnostics. Owing to their size and functional properties, MIP nanoparticles (NPs) can potentially be used in biomedicine, but comprehensive analysis of their interaction with cells and in vitro toxicological tests must be performed first. Herein, we report the synthesis of bare and core–shell imprinted NPs using an innovative solid-phase approach and the toxicological evaluation of such NPs in different cell lines (HaCaT, MEFs, HT1080, and macrophages). We also evaluated the influence of the protein corona on particle stability, the internalization of NPs in cells, and the influence of various surface coatings. Studies on the metabolic effects of imprinted NPs on fibroblasts showed that bare MIPs do not alter cell metabolism, whereas some issues arise when specific particle coatings are used. Furthermore, in vitro cytokine release studies revealed that macrophages were not activated in the presence of the MIPs evaluated in this study. The results suggest that MIP NPs are biocompatible, paving the way for their in vivo application.