Mihaela Puiu

Kinetic approach of aflatoxin B1-acetylcholinesterase interaction: a tool for developing surface plasmon resonance biosensors.

This work presents a kinetic approach of the interaction between acetylcholinesterase (AChE) from electric eel and aflatoxin B1 (AFB1) or its protein conjugate (e.g., AFB1-HRP [horseradish peroxidase]) in order to develop a simple and sensitive detection method of these compounds. The dissociation constant K(d) of the AChE/AFB1-HRP interaction (0.4 μM) obtained with the surface plasmon resonance (SPR) technique is very close to the inhibition constant reported in amperometric assay (K(i)=0.35 μM), proving that the conjugation of AFB1 to a carrier protein does not significantly influence the affinity of AFB1 for AChE. Thus, the AChE/AFB1-HRP couple can be used as mimic system for the binding of AChE to other AFB1-protein adducts and further used for developing biosensors for AFB1 bound to plasma proteins. The immobilization protocol was designed to minimize the nonspecific adsorption on the self-assembled monolayer (SAM) functionalized surface of the SPR chip without an additional hydrophilic linker, whereas the interaction protocol was designed to mark out the possible occurrence of mass transport limitation (MTL) effects. The detection limits (LODs) were 0.008 μM for AFB1-HRP (2.5 ng ml⁻¹ AFB1) and 0.94 ng ml⁻¹ for AFB1 itself, which is lower than recently reported values in spectrophotometric and amperometric assays.

SPR and SPR Imaging: Recent Trends in Developing Nanodevices for Detection and Real-Time Monitoring of Biomolecular Events.

In this paper we review the underlying principles of the surface plasmon resonance (SPR) technique, particularly emphasizing its advantages along with its limitations regarding the ability to discriminate between the specific binding response and the interfering effects from biological samples. While SPR sensors were developed almost three decades, SPR detection is not yet able to reduce the time-consuming steps of the analysis, and is hardly amenable for miniaturized, portable platforms required in point-of-care (POC) testing. Recent advances in near-field optics have emerged, resulting in the development of SPR imaging (SPRi) as a powerful optical, label-free monitoring tool for multiplexed detection and monitoring of biomolecular events. The microarrays design of the SPRi chips incorporating various metallic nanostructures make these optofluidic devices more suitable for diagnosis and near-patient testing than the traditional SPR sensors. The latest developments indicate SPRi detection as being the most promising surface plasmon-based technique fulfilling the demands for implementation in lab-on-a-chip (LOC) technologies.

Oxidase-peroxidase reaction: kinetics of peroxidase-catalysed oxidation of 2-aminophenol

Possible reaction pathways that may lead to horseradish peroxidase inactivation during the aerobic oxidation of 2-aminophenol were investigated using extended kinetic curves. A kinetic model involving the formation of a low-reactive species, Compound III, was proposed and several rate constants were calculated using an optimisation computing program. Sensitivity analysis allowed to conclude that both oxidase and peroxidase cycles occur in 2-aminophenol oxidation.

Enhanced Sensitive Love Wave Surface Acoustic Wave Sensor Designed for Immunoassay Formats

We report a Love wave surface acoustic wave (LW-SAW) immunosensor designed for the detection of high molecular weight targets in liquid samples, amenable also for low molecular targets in surface competition assays. We implemented a label-free interaction protocol similar to other surface plasmon resonance bioassays having the advantage of requiring reduced time analysis. The fabricated LW-SAW sensor supports the detection of the target in the nanomolar range, and can be ultimately incorporated in portable devices, suitable for point-of-care testing (POCT) applications.

Influence of surfactants on the fading of malachite green

The kinetics of the reaction between malachite green (MG) and sodium hydroxide (MG fading) was studied using a spectrophotometric method in the presence of two cationic surfactants, cetyl-benzyl-dimethyl-ammonium chloride (CBDAC) and hexadecyl-trimethylammonium bromide (HTAB) and one anionic surfactant, sodium dodecyl sulphate (SDS) at concentrations below and above critical micellar concentrations. The cationic surfactants have a catalytic effect, while the anionic surfactant has an inhibitory effect on the reaction. A kinetic model describing the influence of surfactant on reaction rate was developed. The results are discussed on the basis of electrostatic and hydrophobic interactions between the kinetic micelles and malachite green.

Estimation of the overall kinetic parameters of enzyme inactivation using an isoconversional method.

An isoconversional method is proposed in order to calculate the kinetic parameters of enzyme inactivation. The method provides an efficient and low-cost procedure to describe both operational and thermal inactivation. Unlike the ordinary kinetic assays performed at constant enzyme concentration and at various substrate concentrations, the isoconversional method requires several extended kinetic curves for constant initial substrate concentration and different enzyme concentrations. The procedure was tested and validated using simulated data obtained for several kinetic models frequently discussed in the literature. After the validation, the isoconversional method was used for the investigation of the thermoinactivation of urease during urea hydrolysis in self buffered medium and the operational inactivation (destructive oxidation by excess peroxide) of catalase at high concentration of hydrogen peroxide. The results showed that the isoconversional method gives good results of global inactivation constant for both simple and more complex models.

Kinetics of hydrogen peroxide decomposition by catalase: hydroxylic solvent effects

The effect of water–alcohol (methanol, ethanol, propan-1-ol, propan-2-ol, ethane-1,2-diol and propane-1,2,3-triol) binary mixtures on the kinetics of hydrogen peroxide decomposition in the presence of bovine liver catalase is investigated. In all solvents, the activity of catalase is smaller than in water. The results are discussed on the basis of a simple kinetic model. The kinetic constants for product formation through enzyme–substrate complex decomposition and for inactivation of catalase are estimated. The organic solvents are characterized by several physical properties: dielectric constant (D), hydrophobicity (log P), concentration of hydroxyl groups ([OH]), polarizability (α), Kamlet-Taft parameter (β) and Kosower parameter (Z). The relationships between the initial rate, kinetic constants and medium properties are analyzed by linear and multiple linear regression.

Temperature and pH effects on the kinetics of 2-aminophenol auto-oxidation in aqueous solution

The kinetics of the auto-oxidation of 2-aminophenol (OAP) to 2-amino-phenoxazin-3-one (APX) was followed in air-saturated aqueous solutions and the influence of temperature and pH on the auto-oxidation rate was studied. The kinetic analysis was based on a spectrophotometric method following the increase of the absorbance of APX. The process follows first order kinetics according to the rate law—d[OAP]/dt=k′[OAP]. The experimental data, within the pH range 4–9.85, were analyzed using both differential and incremental methods. The temperature variation of the overall rate constant was studied at pH=9.85 within the range 25–50°C and the corresponding activation energy was evaluated.

Peptide-based biosensors: From self-assembled interfaces to molecular probes in electrochemical assays

Redox-tagged peptides have emerged as functional materials with multiple applications in the area of sensing and biosensing applications due to their high stability, excellent redox properties and versatility of biomolecular interactions. They allow direct observation of molecular interactions in a wide range of affinity and enzymatic assays and act as electron mediators. Short helical peptides possess the ability to self-assemble in specific configurations with the possibility to develop in highly-ordered, stable 1D, 2D and 3D architectures in a hierarchical controlled manner. We provide here a brief overview of the electrochemical techniques available to study the electron transfer in peptide films with particular interest in developing biosensors with immobilized peptide motifs, for biological and clinical applications.

Permanganate-assisted removal of PCR inhibitors during the DNA Chelex extraction from stained denim samples.

In this study, it was demonstrated that the DNA Chelex extraction combined with the permanganate assisted-oxidation is highly efficient in removing the PCR inhibitors often found in clothing materials, such as phthalocyanine. The extraction assays were conducted in saliva, blood and epithelial cells samples mixed with three oxidation-resistant dye copper(II) α-phthalocyanine, copper(II) β-phthalocyanine and tetrasulfonated copper(II) β-phthalocyanine. After DNA amplification, all samples were able to provide full DNA profiles. The permanganate/Chelex system was tested further on denim-stained samples and displayed the same ability to remove the PCR inhibitors from the commercial textile materials.