New Validated Method for the Determination of Six Opium Alkaloids in Poppy Seed-Containing Bakery Products by High-Performance Liquid Chromatography-Tandem Mass Spectrometry after Magnetic Solid-Phase Extraction.

Bakery products containing poppy seeds are increasingly being commercialized. These seeds may be contaminated with latex from the Papaver somniferum L. plant rich in opium alkaloids (OAs). Therefore, health authorities demand the development of analytical methods to control them. In this study, an efficient and simple method was developed and validated for the first time to analyze six OAs in bakery products by high-performance liquid chromatography-tandem mass spectrometry. For this purpose, a solid-liquid extraction was optimized, and then a magnetic material [magnetite surface-modified with Fe(III) terephthalate, denoted as Fe3O4@TPA-Fe] was used for a fast magnetic solid-phase extraction. The method has been validated with adequate recoveries (70-110%) and relative standard deviations (<20%) and without matrix effects. Nine bakery samples (five breadsticks and four sliced bread) were analyzed; breadsticks showed low amounts of OAs, but two sliced bread showed higher amounts of OAs than the new amount (1.5 mg/kg) set by the Commission Regulation (EU) 2021/2142.

Electrochemical biosensor for the simultaneous determination of rheumatoid factor and anti-cyclic citrullinated peptide antibodies in human serum.

This paper reports a dual electrochemical biosensor involving carboxylated- or neutravidin-functionalized magnetic microbeads and dual screen-printed carbon electrodes for the simultaneous determination of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCPA) autoantibodies used as biomarkers for the detection of rheumatoid arthritis autoimmune disease. Sandwich-type biosensors involving Fc fragments of IgG Fc(IgG) and biotinylated cyclic cytrullinated peptide (CCP-biotin) to form CCP-biotin-Neutr-MBs for the specific immobilization of RF and CCPA, respectively, as well as conjugation with HRP-IgM and HRP-IgG for RF and CCPA, respectively, were prepared. Amperometric detection was performed at -0.20 V vs. Ag pseudo-reference electrode using the H2O2/hydroquinone (HQ) system upon capturing the bioconjugates onto the corresponding working electrode (WE1 or WE2) of SPCdEs. The dual biosensor exhibits high sensitivity for RF and CCPA with LOD values of 0.8 and 2.5 IU mL-1, respectively. The simultaneous determination can be completed in about two hours using a simple protocol and a sample volume (25 µL) four times smaller than that required by the ELISA method. The dual electrochemical biosensor was used for the determination of both target biomarkers in human serum.

Dendrimers as Soft Nanomaterials for Electrochemical Immunosensors.

Electrochemical immunosensors are antibody-based affinity biosensors with a high impact on clinical, environmental, food, and pharmaceutical analysis. In general, the analytical performance of these devices is critically determined by the materials and reagents used for their construction, signal production and amplification. Dendrimers are monodisperse and highly branched polymers with three-dimensional structures widely employed as "soft" nanomaterials in electrochemical immunosensor technology. This review provides an overview on the state-of-the-art in dendrimer-based electrochemical immunosensors, focusing on those using polyamidoamine and poly (propylene imine) dendrimers. Special emphasis is given to the most original methods recently reported for the construction of immunosensor architectures incorporating dendrimers, as well as to novel sensing approaches based on dendrimer-assisted signal enhancement strategies.

Amperometric immunoassay for the obesity biomarker amylin using a screen printed carbon electrode functionalized with an electropolymerized carboxylated polypyrrole.

Amylin (the islet amyloid polypeptide) is a hormone related to adiposity, hunger and satiety. It is co-secreted with insulin from pancreatic B-cells. An amperometric immunosensor is presented here for the determination of amylin. It is making use of a screen printed carbon electrode (SPCE) functionalized with electropolymerized poly(pyrrole propionic acid) (pPPA) with abundant carboxyl groups that facilitate covalent binding of antibody against amylin. A competitive immunoassay was implemented using biotinylated amylin and streptavidin labeled with horse radish peroxidase (HRP-Strept) as the enzymatic tracer. The amperometric detection of H2O2 mediated by hydroquinone was employed as an electrochemical probe to monitor the affinity reaction. The variables involved in the preparation and function of the immunosensor were optimized and the electrodes were characterized by electrochemical impedance spectroscopy and cyclic voltammetry. The calibration graph for amylin, obtained by amperometry at -200 mV vs Ag pseudo-reference electrode, showed a range of linearity extending from 1.0 fg∙mL-1 to 50 pg∙mL-1, with a detection limit of 0.92 fg∙mL-1. This is approximately 7000 times lower than the minimum detectable concentration reported for the ELISA immunoassays available for amylin. The assay has excellent reproducibility and good selectivity over potential interferents. Graphical abstract Schematic of an amperometric competitive immunoassay for the obesity biomarker amylin using a poly(pyrrole propionic acid)-modified screen-printed electrode. The detection limit is 0.92 fg∙mL-1 amylin. The method provides excellent reproducibility for the measurements, good selectivity and successful applicability to human urine and serum samples.

An Electrochemical Enzyme Biosensor for 3-Hydroxybutyrate Detection Using Screen-Printed Electrodes Modified by Reduced Graphene Oxide and Thionine.

A biosensor for 3-hydroxybutyrate (3-HB) involving immobilization of the enzyme 3-hydroxybutyrate dehydrogenase onto a screen-printed carbon electrode modified with reduced graphene oxide (GO) and thionine (THI) is reported here. After addition of 3-hydroxybutyrate or the sample in the presence of NAD⁺ cofactor, the generated NADH could be detected amperometrically at 0.0 V vs. Ag pseudo reference electrode. Under the optimized experimental conditions, a calibration plot for 3-HB was constructed showing a wide linear range between 0.010 and 0.400 mM 3-HB which covers the clinically relevant levels for diluted serum samples. In addition, a limit of detection of 1.0 µM, much lower than that reported using other biosensors, was achieved. The analytical usefulness of the developed biosensor was demonstrated via application to spiked serum samples.

Electrochemical immunosensor for ethinylestradiol using diazonium salt grafting onto silver nanoparticles-silica-graphene oxide hybrids.

This work describes the preparation of an electrochemical immunosensor for ethinylestradiol (EE2) based on grafting of diazonium salt of 4-aminobenzoic acid onto a glassy carbon electrode modified with silver nanoparticles/SiO2/graphene oxide hybrid followed by covalent binding of anti-ethinylestradiol (anti-EE2) to activated carboxyl groups. A competitive immunoassay was developed for the determination of the hormone using peroxidase-labeled ethinylestradiol (HRP-EE2) and measurement of the amperometric response at -200mV in the presence of hydroquinone (HQ) as redox mediator. The calibration curve for EE2 exhibited a linear range between 0.1 and 50ng/mL (r(2)=0.996), with a detection limit of 65pg/mL. Interference studies with other hormones related with EE2 revealed the practical specificity of the developed method for the analyte. A good reproducibility, with RSD=4.5% (n=10) was also observed. The operating stability of a single bioelectrode modified with anti-EE2 was maintained at least for 15 days when it was stored at 4°C under humid conditions between measurements. The developed immunosensor was applied to the analysis of spiked urine with good results.