Francesca Bettazzi

Disposable electrochemical DNA-array for PCR amplified detection of hazelnut allergens in foodstuffs

An electrochemical low-density DNA-array has been designed and implemented to be used in combination with polymerase chain reaction (PCR) in order to investigate the presence of hazelnut major allergens (Cor a 1.04, Cor a 1.03) in foodstuff. Unmodified PCR products were captured at the sensor interface via sandwich hybridization with surface-tethered probes and biotinylated signalling probes. The resulting biotinylated hybrids were coupled with a streptavidin-alkaline phosphatase conjugate and then exposed to a alpha-naphthyl phosphate solution. Differential pulse voltammetry was finally used to detect the alpha-naphthol signal. The detection limits for Cor a 1.03 and Cor a 1.04 were 0.3 and 0.1 nmol L(-1), respectively (R.S.D. 10%). The optimized conditions were used to test several commercially available foodstuffs, claiming to contain or not the targeted nuts. The results were compared with those obtained with classical ELISA tests.

Electrochemical bioassay for the detection of TNF-α using magnetic beads and disposable screen-printed array of electrodes

BACKGROUND In this study we have developed an electrochemical bioassay for the analysis of TNF-α, coupling magnetic beads with disposable electrochemical platforms. TNF-α is a pro inflammatory cytokine that participates in the regulation of immune defense against various pathogens and the recovery from injury. It plays a central role in the development of many inflammatory diseases. The bioassay was based on a sandwich format using alkaline phosphatase as an enzymatic label and an eight-sensor screen-printed array as an electrochemical transducer. RESULTS The modified magnetic beads were captured by a magnet on the surface of each graphite working electrode of the array and the electrochemical detection was thus achieved through the addition of the alkaline phosphatase substrate (1-naphthylphosphate); 1-naphthol produced during the enzymatic reaction was detected using differential pulse voltammetry. The parameters influencing the different steps of the assay were optimized in order to reach the best sensitivity and specificity. CONCLUSION The proposed strategy offers great promise for analysis of clinical diagnostics, considering also that arrays allow the simultaneous analysis of different samples.

Strategies for the development of an electrochemical bioassay for TNF-alpha detection by using a non-immunoglobulin bioreceptor.

TNF-α is an inflammatory cytokine produced by the immune system. Serum TNF-α level is elevated in some pathological states such as septic shock, graft rejection, HIV infection, neurodegenerative diseases, rheumatoid arthritis and cancer. Detecting trace amount of TNF-α is, also, very important for the understanding of tumor biological processes. Detection of this key biomarker is commonly achieved by use of ELISA or cytofluorimetric based methods. In this study the traditional optical detection was replaced by differential pulse voltammetry (DPV) and an affinity molecule, produced by evolutionary approaches, has been tested as capture bioreceptor. This molecule, namely a combinatorial non-immunoglobulin protein (Affibody®) interacts with TNF-α selectively and was here tested in a sandwich assay format. Moreover magnetic beads were used as support for bioreceptor immobilization and screen printed carbon electrodes were used as transducers. TNF-α calibration curve was performed, obtaining the detection limit of 38pg/mL, the quantification range of 76-5000pg/mL and RSD%=7. Preliminary results of serum samples analysis were also reported.

Direct determination of small RNAs using a biotinylated polythiophene impedimetric genosensor

Herein, direct determination of small RNAs is described using a functional-polymer modified genosensor. The analytical strategy adopted involves deposition by electropolymerization of biotinylated polythiophene films on the surface of miniaturized, disposable, gold screen-printed electrodes, followed by the layer-by-layer deposition of streptavidin, and then biotynilated capture probes. A small RNA (miR-221) target was determined via the impedimetric measurement of the hybridization event in a label-free and PCR-free approach. Under optimized conditions, the limit of detection (LOD) was 0.7 pM miR-221 (15% RSD). The genosensor was applied for determination of miR-221 in total RNA extracted from human lung and breast cancer cell lines, discriminating between the cancer-positive and -negative cells, without any amplification step, in less than 2h.

Health and carcinogenic risk evaluation for cohorts exposed to PAHs in petrochemical workplaces in Rawalpindi city (Pakistan).

This study presents the analyses of urinary biomarkers (1-OHPyr, α- and β-naphthols) of polycyclic aromatic hydrocarbons (PAHs) exposure and biomarkers of effect (i.e. blood parameters) in petroleum-refinery workers (RFs) and auto-repair workers (MCs). Exposed subjects had higher concentrations of white blood cell (WBC) count than control subjects (CN) subjects (5.31 × 103 μL−1 in exposed vs. 5.15 × 103 μL−1 in CN subjects), while the biomarker of oxidative DNA damage (8-OHdG) was significantly higher in MCs. The exposure among these two cohorts could be influenced by the ambience of the workplaces; in fact, MCs’ shops are relatively damp and enclosed workplaces in comparison with the indoor environment of refineries. PAHs in the dust samples from mechanical workshops probably originated from mixed sources (traffic exhaust and petroleum spills), while the incremental lifetime cancer risk (ILCR) for MCs showed moderate-to-low cancer risk from exposure to dust-bound PAHs. The study shows that increasing PAH exposure can be traced in MC workstations and needs to be investigated for the safety of public health.

Imidazo[1,2-a]pyrazin-8-amine core for the design of new adenosine receptor antagonists: Structural exploration to target the A3 and A2A subtypes

The imidazo[1,2-a]pyrazine ring system has been chosen as a new decorable core skeleton for the design of novel adenosine receptor (AR) antagonists targeting either the human (h) A3 or the hA2A receptor subtype. The N8-(hetero)arylcarboxyamido substituted compounds 4-14 and 21-30, bearing a 6-phenyl moiety or not, respectively, show good hA3 receptor affinity and selectivity versus the other ARs. In contrast, the 8-amino-6-(hetero)aryl substituted derivatives designed for targeting the hA2A receptor subtype (compounds 31-38) and also the 6-phenyl analogues 18-20 do not bind the hA2A AR, or show hA1 or balanced hA1/hA2A AR affinity in the micromolar range. Molecular docking of the new hA3 antagonists was carried out to depict their hypothetical binding mode to our refined model of the hA3 receptor. Some derivatives were evaluated for their fluorescent potentiality and showed some fluorescent emission properties. One of the most active hA3 antagonists herein reported, i.e. the 2,6-diphenyl-8-(3-pyridoylamino)imidazo[1,2-a]pyrazine 29, tested in a rat model of cerebral ischemia, delayed the occurrence of anoxic depolarization caused by oxygen and glucose deprivation in the hippocampus and allowed disrupted synaptic activity to recover.

Electrochemical Liposome-Based Biosensors for Nucleic Acid Detection

MicroRNAs (miRNAs) are intensely studied as candidates for diagnostic and prognostic clinical biomarkers. Faradic impedance spectroscopy (EIS) and differential pulse voltammetry (DPV), coupled to disposable gold electrodes and enzyme amplification of the hybridization event, were used for the development of biosensors for the detection of miRNAs. Biotin-labeled liposomes were employed as nanointerfaces that amplify the primary miRNA-sensing events by their association to the probe–/DNA–miRNA–analyte complex generated onto the transducer.

Evaluation of a QuEChERS-like extraction approach for the determination of PBDEs in mussels by immuno-assay-based screening methods

A sample preparation method was evaluated for the determination of polybrominated diphenyl ethers (PBDEs) in mussel samples, by using colorimetric and electrochemical immunoassay-based screening methods. Herein, a rapid procedure based on QuEChERS-like extraction approach followed by solid phase purification was optimized for PBDE extraction from mussel samples. The detection limits for colorimetric and electrochemical immunoassays, calculated as BDE-47 equivalent concentration, were 0.6ngg-1 and 1.1ngg-1, respectively. Real mussel samples, including a Certified Reference Material (CRM), were analyzed. The samples were measured by colorimetric and electrochemical immunoassays as well as by GC-MS. In comparison to GC-MS results, 106% and 102% relative accuracy were obtained for the colorimetric and electrochemical immunoassays, respectively. The proposed method could be useful for massive environmental campaigns, being able to rapidly detect possible polluted seafood samples.

Macrocyclic Polyamine Modified Screen-Printed Electrodes for Copper(II) Detection

The development of a mercury-free electrochemical sensor for the detection of Cu(II) is described. In particular screen-printed carbon-based electrodes were modified with a membrane constituted by Nafion™ containing a macrocyclic polyamine derivative. The “medium exchange technique” was used to develop a procedure of accumulation of metal ions followed by square wave anodic stripping voltammetry (SWASV).

Glyphosate Determination by Coupling an Immuno-Magnetic Assay with Electrochemical Sensors

Glyphosate (N-(phosphonomethyl)glycine) is the most frequently used broad-spectrum herbicide worldwide. Its mechanism of action is based on the inhibition of an enzyme that is essential to plant growth. Its intensive use has caused global contamination to occur, which has not only affected the ecosystems, but even food and other objects of common use. Thus, there is a pronounced need for developing analytical methods for glyphosate determination in different matrices. Here, an electrochemical competitive immunoassay, based on the use of antibody-modified magnetic particles, has been developed. Tetramethylbenzidine (TMB) has been used as an enzymatic substrate. The extent of the affinity reaction has been achieved by monitoring the current value, due to the reduction of the enzymatic product. A disposable screen-printed electrochemical cell has been used. The calibration curve has been recorded in the 0–10,000 ng/L concentration range, with a detection limit of 5 ng/L and quantification limit of 30 ng/L. The electrochemical immunoassay has also been applied to the analysis of spiked beer samples.