Dimitrios P. Nikolelis

Biosensors Based on Thin Lipid Films and Liposomes

This article reports the theory and analytical applications of thin lipid films. Recent advances of electrochemical devices based on lipid membranes have lead to reports of construction of biosensors for environmental and food applications, and may provide opportunities for commercial fabrication. The methods of formation of lipid membranes on various supports including metals (silver, gold, stainless steel), agar, conducting polymers and ultrafiltration membranes have provided stabilization of lipid films with a diversity of analytical applications in real samples. Methods of immobilization and incorporation of various functional macromolecules are summarized. Several examples of the application of various methods for study of physical properties of supported bilayer lipid membranes are described. Applications of lipid-based biosensors in analytical chemistry for determination of compounds are demonstrated, including a diversity of chemical compounds such as environmental pollutants (ammonia and carbon dioxide, cyanide ions, etc.) and food toxins (aflatoxin M1and direct detection of toxin in real samples such as milk and milk preparations). Methods for application of liposomes as a sensing system are also summarized.

Pre-concentration of indolic compounds at a carbon paste electrode and indirect determination of L-tryptophan in serum by adsorptive stripping voltammetry

The pre-concentration of indole and other indolic compounds of biochemical and pharmaceutical interest at a carbon paste electrode (Nujol/graphite) has been studied. All the compounds examined can be determined, by direct voltammetric measurements in their solutions, in the concentration range 1-8 microM. Indole, methylated indoles, harmaline and serotonin were accumulated at the electrode by a combined adsorption/extraction process. By applying the medium-exchange procedure, the accumulated compounds can be determined in the same concentration range, after a 60-s pre-concentration period, enhancing, therefore, the selectivity of the voltammetric determination. A procedure for the indirect determination of L-tryptophan in serum has been developed, which is based on these findings. L-Tryptophan was cleaved to indole by tryptophanase, and indole was subsequently determined voltammetrically after a 2-min pre-concentration period at the carbon paste electrode. From 0.3 to 1.2 micrograms of L-tryptophan in a total of 75 microliters of serum sample (20-80 microM) can be determined with an average error of ca. 0.03 microgram, whereas the recovery of added L-tryptophan in serum samples is in the range 105-115%.

Construction of an arginine enzyme electrode and determination of arginine in biological materials

The arginine electrode is based on a coupled enzymatic system consisting of arginase and urease with an ammonia gas sensor; conditions of immobilization are optimized. Arginine in the range 3 × 10−5–3 × 10−3 M gives a linear potential vs. log (concentration) plot with a response time of 5 min over the range specified. Several compounds structurally related to arginine do not interfere. The method is suitable for the determination of arginine in bovine insulin and human serum. Results compare well with values given in the literature or obtained by the conventional Sakaguchi method.

Direct electrochemical transduction of an immunological reaction by bilayer lipid membranes

This work reports the electrochemical transduction of an immunological interaction by use of bilayer lipid membranes (BLMs)which were prepared from mixtures of egg phosphatidylcholine (PC) and dipalmitoyl phosphatidic acid (DPPA). Thyroxin (T4)/anti-rabbit T4 was used as a representative immunological reaction for these studies. Antibody-antigen complexation caused transient ion current signals due to dynamic changes of the electrostatic fields at the surface of such membranes. The mechanism of signal generation is based on the perturbation of the electrical double layer and surface structure of the BLMs. The transient charging signals occurred as singular or multiple events which lasted for a period on the order of seconds. The magnitude of these transient ion current signals was directly related to the concentration of the antigen in bulk solution, which could be determined over a range of nM to mM levels in a period of seconds to minutes. Investigation of the effects of lipid composition of BLMs, pH and the presence of Ca2+_ in bulk electrolyte solution indicated that the response could be optimized for sensitivity and speed.

Electrochemical transduction of the acetylcholine-acetylcholinesterase reaction by bilayer lipid membranes

Abstract This work reports the transduction of the reaction of the enzyme acetylcholinesterase (AChE) with acetylcholine (ACh) as a model to demonstrate how transient electrochemical signals from bilayer lipid membranes can be obtained by appropriate selection of the lipid composition of membranes. Membranes were prepared from mixtures of egg phosphatidyl choline (PC) and dipalmitoyl phosphatidic acid (DPPA) for this purpose. Hydronium ions generated by the enzymatic reaction at the surface of BLMs caused a transient current due to a dynamic alteration of the electrostatic fields at the surface of such membranes. The results were consistent with an electrostatic mechanism of perturbation of the surface structure of the BLMs, where changes of local hydronium ion activity which were associated with the enzymatic altered the extent of ionization of the headgroups of the DPPA, thereby providing a transient charging current which lasted for a period on the order of seconds. The delay time for observation of the transient was directly and reproducibly related to the concentration of the substrate, which could be determined over a range of μM to mM levels. Investigation of the effects of solution pH, the presence of Ca 2+ and the use of the enzyme inhibitor Neostigmine confirmed that the response was due to a genuine selective chemical transduction process.

Biosensors for the Rapid Detection of Dopamine Using Bilayer Lipid Membranes (BLMs) With Incorporated Calix[4]resorcinarene Receptor

This work explores the use of a calix[4]resorcinarene receptor (lipophilic macrocyclic host molecule) for the rapid electrochemical detection of dopamine using planar bilayer lipid membranes (BLMs). BLMs were composed of egg phosphatidylcholine (PC) and 35% (w/w) dipalmitoyl phosphatidic acid in which the receptor was incorporated. Freely-suspended and metal supported BLMs modified with the resorcin[4]arene receptor were used as one shot sensors to rapidly detect this catecholamine. The interactions of this compound with freely-suspended BLMs were found to be electrochemically transduced in the form of a transient current signal with duration of seconds, which reproducibly appeared within 8 s after exposure of the membranes to dopamine. The response time for these BLMs without incorporated receptor for dopamine was about 3 min. The magnitude of the transient current signal was related to the concentration of the stimulating agent in bulk solution in the micromolar range. The present electrochemical technique was also applied for the rapid and sensitive screening of dopamine using surface-stabilized bilayer lipid membranes (sBLMs). The interactions of dopamine with sBLMs with incorporated receptor produced electrochemical ion current increases, which reproducibly appeared within a few seconds after exposure of the membranes to the stimulant. The use of the receptor in sBLMs increased the sensitivity of the method by 10-fold. The current signal increases were related to the concentration of dopamine in bulk solution in the micromolar range. No interferences from ascorbic acid were noticed because of the use of the negatively charged lipids in membranes. The present technique can be used as one-shot sensor for the rapid detection of these pharmaceutical substances and keep prospects for the selective determination of catecholamines in biofluids.

Flow Injection Monitoring of Aflatoxin M1 in Cheese Using Filter-Supported Bilayer Lipid Membranes with Incorporated DNA

This work describes a technique for the rapid and sensitive electrochemical flow injection monitoring of aflatoxin M1 (AFM1) in cheese samples. Stabilized filter-supported bilayer lipid membranes (BLMs) were used as detectors Single stranded DNA oligomers terminated with alkyl chains (dT20–C16) were incorporated into the membranes to control surface electrostatic properties. The incorporation of dT20–C16 in BLMs lowered the detection limit for the detection of this toxin by one to four orders of magnitude as compared with the detection limit obtained in the absence of DNA. Therefore, it is now possible to continuously monitor this toxin at concentrations that approached those that could be of interest as set by the U.S. Food and Drug Administration and most European countries. The work described herein takes a significant step towards development of a detector of greater practical potential by demonstrating that the incorporation of C16-ssDNA into lipid membranes results in a combination of properties that provides for a much more sensitive and robust detection system. Injections of AFM1 were made into flowing streams of a 0.1 M KCl electrolyte solution, and a transient current signal with duration of seconds reproducibly appeared in about 12 s after exposure of the detector element to the toxin. The magnitude of this signal was linearly related to the concentration of AFM1 with detection limits at subnanomolar level. The effect of interferents such as proteins and lipids was investigated. It was determined that interferences from proteins could be eliminated by adjustment of the flow rate of the carrier electrolyte solution. The technique was applied for the rapid flow injection determination of aflatoxin M1 in cheese samples. AFM1 could be determined in continuous flowing systems with a rate of at least 4 samples min–1. Repetitive cycles of injection of AFM1 have shown no signal degradation during each cycle for experiments that attempted over 30 cycles of detection.

A minisensor for the rapid screening of sucralose based on surface-stabilized bilayer lipid membranes

This work describes an electrochemical technique that is suitable for the rapid and sensitive screening of the sweetener sucralose based on surface-stabilized bilayer lipid membranes (s-BLMs) composed of egg phosphatidylcholine. The interactions of sucralose with s-BLMs produced electrochemical ion current increases, which appeared reproducible within a few seconds after exposure of the membranes to the sweetener. The mechanism of signal generation was investigated by differential scanning calorimetric studies. The mechanism was found to be associated with alteration of the electrostatic fields of the lipid film. These studies revealed that an increase of the molecular area of the lipids at the membranes and a stabilization of a gel phase structure occurred due to adsorption of the sweetener. Water molecules are adsorbed at the polar headgroups of the lipids, which changes the electrostatic field at the surface of the membranes. The current signal increases were related to the concentration of sucralose in bulk solution in the micromolar range. The present lipid film based sensor provided a fast response (i.e. in the order of a few seconds) to alterations of sucralose concentration (5-50 microm) in electrolyte solution. The electrochemical transduction of the interactions of this artificial sweetener with s-BLMs was applied in the determination of this compound in granulated sugar substitute products using the present minisensor.

Direct electrochemical sensing of insecticides by bilayer lipid membranes

Abstract This work describes the use of bilayer lipid membranes (BLMs) as sensitive detectors for the direct electrochemical monitoring of the organophosphate and carbamate insecticides monocrotofos and carbofuran, respectively. Egg phosphatidylcholine (PC) and dipalmitoylphosphatidic acid (DPPA) were used for the formation of BLMs. The interactions of monocrotofos and carbofuran with BLMs produced a transient current signal with a duration of seconds, which reproducibly appeared within 3 to 5 min after exposure of the membranes to the insecticides. The sensitivity of response was maximized by use of high concentrations of the charged lipid, and by alteration of the phase distribution within membranes by the introduction of calcium ions in bulk solution. The mechanism of signal generation is related to the absorption of the lipophilic insecticide molecules with a consequent rapid reorganization of the membrane electrostatics. The magnitude of the transient current signal was linearly related to the concentration of monocrotofos or carbofuran in bulk solution with sub-micromolar detection limits.

Investigation of interactions of a resorcin[4]arene receptor with bilayer lipid membranes (BLMs) for the electrochemical biosensing of mixtures of dopamine and ephedrine.

The present article investigates the interactions of a resorcin[4]arene receptor with planar bilayer lipid membranes (BLMs) that can be used for the electrochemical detection of dopamine and ephedrine. BLMs were composed of egg phosphatidylcholine and 35% (w/w) dipalmitoyl phosphatidic acid in which the receptor was incorporated. These BLMs modified with the resorcin[4]arene receptor can be used as one-shot sensors for the direct electrochemical sensing of these energizing-stimulating substances. The interactions of these compounds with the lipid membranes were found to be electrochemically transduced in the form of a transient current signal with a duration of seconds, which reproducibly appeared within 8 and 20 s after exposure of the membranes to dopamine and ephedrine, respectively. The response time for BLMs without the receptor for dopamine was about 3 min, whereas no signals were obtained for ephedrine in the absence of the receptor. The mechanism of signal generation was investigated by differential scanning calorimetric studies. These studies revealed that the adsorption of the receptor is through the hydrophobic tails of the receptor, whereas hydrophilic groups of the receptor were directed towards the electrolyte solution enhancing the ion transport through the lipid membranes. The magnitude of the transient current signal was related to the concentration of the stimulating agent in bulk solution in the micromolar range. No interferences from ascorbic acid were noticed because of the use of the negatively charged lipids in membranes. The present technique can be used as one-shot sensor for the detection of these pharmaceutical substances and future research is targeted to the determination of these chemicals in human biofluids such as urine of athletes.