Lucian Rotariu

A novel, sensitive, reusable and low potential acetylcholinesterase biosensor for chlorpyrifos based on 1-butyl-3-methylimidazolium tetrafluoroborate/multiwalled carbon nanotubes gel.

A novel, low potential and highly sensitive acetylcholinesterase (AChE) biosensor was developed based on 1-butyl-3-methylimidazolium tetrafluoroborate/multiwalled carbon nanotube composite gel thiocholine sensor. Composite gel promoted electron transfer reaction at a lower potential (+50 mV) and catalyzed electrochemical oxidation of thiocholine with high sensitivity. AChE was immobilized in sol-gel matrix that provides a good support for enzyme without any inhibition effect from the ionic liquid. The amount of immobilized enzyme and incubation time with chlorpyrifos were optimized. Chlorpyrifos could be determined in the range of 10(-8)-10(-6)M with a detection limit of 4 nM. Fast and efficient enzyme reactivation was obtained at low obidoxime concentration (0.1mM). Moreover, the biosensor exhibited a good stability and reproducibility and could be use for multiple determinations of pesticide with no loss of the enzyme activity.

A novel amperometric biosensor based on gold nanoparticles anchored on reduced graphene oxide for sensitive detection of l-lactate tumor biomarker

In this work, a novel amperometric biosensor based on gold nanoparticles anchored on reduced graphene oxide (RGO-AuNPs) and l-lactate dehydrogenase (LDH) was developed for the sensing of l-lactate. Firstly, the RGO-AuNPs modified screen printed electrodes were tested for NADH detection showing a wide dynamic range and a low detection limit. Next, the biosensor was constructed by incorporating both enzyme and RGO-AuNPs in a sol gel matrix derived from tetrametoxysilane and methyltrimetoxysilane. The enzyme loading, working pH, and coenzyme concentration were optimized. The biosensor linearly responded to l-lactate in the range of 10µM-5mM and showed a good specific sensitivity of 154µA/mMcm(2) with a detection limit of 0.13µM. This was accompanied by good reproducibility and operational stability. Tests on artificial serum proved that l-lactate can be determined practically without interferences from commonly interfering compounds such as urate, paracetamol and l-ascorbate. Our LDH/RGO-AuNPs/SPCE based biosensor thus performs as electrochemical device for the detection of l-lactate as a viable early cancer bio-marker.

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.

A rational design of the multiwalled carbon nanotube-7,7,8,8-tetracyanoquinodimethan sensor for sensitive detection of acetylcholinesterase inhibitors.

A new, simple and effective amperometric acetylcholinesterase biosensor was developed using screen-printed carbon electrodes modified with carbon nanotubes (MWCNTs)-7,7,8,8-tetracyanoquinodimethane (TCNQ). The design of the biosensor was based on the supramolecular arrangement resulted from the interaction of MWCNTs and TCNQ. This arrangement was confirmed by spectroscopic and electrochemical techniques. Two different supramolecular arrangements were proposed based on different MWCNTs:TCNQ ratios. The synergistic effect of MWCNTs and TCNQ was, for the first time, exploited for detection of thiocholine at low potential with high sensitivity. The biosensor developed by immobilization of acetylcholinesterase (AChE) in sol-gel allowed the detection of two reference AChE inhibitors, paraoxon-methyl and chlorpyrifos with detection limits of 30 pM (7 ppt) and 0.4 nM (0.1 ppb), respectively. Efficient enzyme reactivation was obtained by using obidoxime.

Sensitive detection of endocrine disrupters using ionic liquid--single walled carbon nanotubes modified screen-printed based biosensors.

Simple and low cost biosensor based on screen-printed electrode for sensitive detection of some alkylphenols was developed, by entrapment of HRP in a nanocomposite gel based on single-walled carbon nanotubes (SWCNTs) and 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF(6)]) ionic liquid. Raman and FTIR spectroscopy, CV and EIS studies demonstrate the interaction between SWCNTs and ionic liquid. The nanocomposite gel, SWCNT-[BMIM][PF(6)] provides to the modified sensor a considerable enhanced electrocatalytic activity toward hydrogen peroxide reduction. The HRP based biosensor exhibits high sensitivity and good stability, allowing a detection of the alkylphenols at an applied potential of -0.2V vs. Ag/AgCl, in linear range from 5.5 to 97.7 μM for 4-t-octylphenol and respectively, between 5.5 and 140 μM for 4-n-nonylphenol, with a response time of about 5s. The detection limit was 1.1 μM for 4-t-octylphenol, and respectively 0.4 μM for 4-n-nonylphenol (S/N=3).

Acetylcholinesterase biosensor for carbamate drugs based on tetrathiafulvalene-tetracyanoquinodimethane/ionic liquid conductive gels.

A highly sensitive acetylcholinesterase biosensor was developed for detection of carbamate drugs based on TTF-TCNQ-ionic liquid gel thiocholine sensor. The TTF-TCNQ-ionic/ionic liquid gel was characterized by FT-IR and scanning electron microscopy. The electrocatalytic behavior of TTF-TCNQ-ionic liquid gels toward oxidation of thiocholine was thoroughly investigated. 1-Ethyl-3-methylimidazolium tetracyanoborate gel based sensor allowed amperometric detection of thiocholine at +400 mV vs. Ag/AgCl with a high sensitivity of 55.9±1.2 μA mM(-1)cm(-2) and a low detection limit equal to 7.6 μM. The catalytic rate constant and diffusion constant of thiocholine were estimated from chronoamperometric data. The proposed biosensor based on AChE immobilized in sol-gel matrix was used for the detection of two carbamate therapeutic drugs. Very low detection limits of 26 pM eserine and 0.3 nM neostigmine were achieved. The analysis of spiked tap water proved the biosensor capability to be used as a screening method for detection of carbamate drugs in wastewaters.

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.

New Type of Ethanol Microbial Biosensor Based on a Highly Sensitive Amperometric Oxygen Electrode and Yeast Cells

Abstract A microbial biosensor for ethanol determination was prepared by immobilization of yeast cells of Saccharomyces ellipsoideus on the surface of an oxygen electrode with electrolyte in nonaqueous medium. Analytical determination is based on the respiratory activity of the microorganism in presence of the analyte. Response time of approximately 5 min for steady-state method and 2 min for kinetic method was registered. A lower detection limit of 6 μM was achieved comparing with previously reported biosensor. The major interfering compound in ethanol assay was glucose. Use of a second PTFE membrane to cover the biocatalytical yeast layer allowed developing a highly selective biosensor for ethanol.

Synergistic effect of mediator-carbon nanotube composites for dehydrogenases and peroxidases based biosensors.

Very sensitive, low cost and reliable NADH and H(2)O(2) sensors were realised and used for development of enzyme based biosensors. The active surface of the electrodes was modified with a nanocomposite obtained by modification of SWNT with a proper mediator: Meldola Blue (for NADH) and Prussian Blue (for H(2)O(2)). Low applied potential of -50 mV vs. Ag/AgCl reference electrode proved the synergistic effect of nanocomposite materials towards NADH and H(2)O(2) detection. Biosensors for malic acid and alkylphenols have been developed, using mediator-functionalized-SWNT-based electrodes and two different classes of enzymes: NAD(+)-dependent dehydrogenases and peroxidases. Immobilization of the enzymes was realised using a series of different procedures - adsorption, Nafion membrane, sol-gel and glutaraldehyde, in order to find the best configuration for a good operational stability. A higher sensitivity comparing with other reported biosensors of about 12.41 mA/M.cm(2) was obtained for l-malic acid biosensor with enzyme immobilised in Nafion membrane. Phenol, 4-t-octylphenol and 4-n-nonylphenol were used as standard compounds for HRP based biosensor. Fast biosensor response and comparable detection limit with HPLC methods were achieved.

DEVELOPMENT OF A PESTICIDES BIOSENSOR USING CARBON-BASED ELECTRODE SYSTEMS

The biosensors described in this work, for the monitoring of pesticides, are based on acetylcholinesterase immobilized on the surface of screen-printed electrodes. The principle of the biosensor is that the degree of inhibition of an enzyme sensor by a pesticide is dependent on the concentration of that pesticide. The DPV technique was used as a detection method and methyl-paraoxon as a reference pesticide for sensor calibration.