Label-Free and Redox Markers-Based Electrochemical Aptasensors for Aflatoxin M1 Detection.

We performed a comparative analysis of the sensitivity of aptamer-based biosensors for detection mycotoxin aflatoxin M1 (AFM1) depending on the method of immobilization of DNA aptamers and method of the detection. Label-free electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) for ferrocene labeled neutravidin layers were used for this purpose. Amino-modified DNA aptamers have been immobilized at the surface of polyamidoamine dendrimers (PAMAM) of fourth generation (G4) or biotin-modified aptamers were immobilized at the neutravidin layer chemisorbed at gold surface. In the first case the limit of detection (LOD) has been determined as 8.47 ng/L. In the second approach the LOD was similar 8.62 ng/L, which is below of allowable limits of AFM1 in milk and milk products. The aptasensors were validated in a spiked milk samples with good recovery better than 78%. Comparative analysis of the sensitivity of immuno- and aptasensors was also performed and showed comparable sensitivity.

The Application of Lipid Membranes in Biosensing.

The exploitation of lipid membranes in biosensors has provided the ability to reconstitute a considerable part of their functionality to detect trace of food toxicants and environmental pollutants. This paper reviews recent progress in biosensor technologies based on lipid membranes suitable for food quality monitoring and environmental applications. Numerous biosensing applications based on lipid membrane biosensors are presented, putting emphasis on novel systems, new sensing techniques, and nanotechnology-based transduction schemes. The range of analytes that can be currently using these lipid film devices that can be detected include, insecticides, pesticides, herbicides, metals, toxins, antibiotics, microorganisms, hormones, dioxins, etc. Technology limitations and future prospects are discussed, focused on the evaluation/validation and eventually commercialization of the proposed lipid membrane-based biosensors.

Application of Biosensors Based on Lipid Membranes for the Rapid Detection of Toxins.

Lipid assemblies in the form of two dimensional films have been used extensively as biosensing platforms. These films exhibit certain similarities with cell membranes, thus providing a suitable means for the immobilization of proteinaceous moieties and, further, a number of intrinsic signal amplification mechanisms. Their implementation in the detection of toxins yielded reliable and fast detectors for in field analyses of environmental and clinical samples. Some examples are presented herein, including aflatoxin and cholera toxin detection. The conditions and parameters that determine the analytical specifications of the lipid membrane sensors are discussed, advantages and technology bottlenecks are reviewed, and possible further developments are highlighted.

Novel Biosensors for the Rapid Detection of Toxicants in Foods.

The modern environmental and food analysis requires sensitive, accurate, and rapid methods. The growing field of biosensors represents an answer to this demand. Unfortunately, most biosensor systems have been tested only on distilled water or buffered solutions, although applications to real samples are increasingly appearing in recent years. In this context, biosensors for potential food applications continue to show advances in areas such as genetic modification of enzymes and microorganisms, improvement of recognition element immobilization, and sensor interfaces. This chapter investigates the progress in the development of biosensors for the rapid detection of food toxicants for online applications. Recent progress in nanotechnology has produced affordable, mass-produced devices, and to integrate these into components and systems (including portable ones) for mass market applications for food toxicants monitoring. Sensing includes chemical and microbiological food toxicants, such as toxins, insecticides, pesticides, herbicides, microorganisms, bacteria, viruses and other microorganisms, phenolic compounds, allergens, genetically modified foods, hormones, dioxins, etc. Therefore, the state of the art of recent advances and future targets in the development of biosensors for food monitoring is summarized as follows: biosensors for food analysis will be highly sensitive, selective, rapidly responding, real time, massively parallel, with no or minimum sample preparation, and platform suited to portable and handheld nanosensors for the rapid detection of food toxicants for online uses even by nonskilled personnel.

Lipid Membrane Nanosensors for Environmental Monitoring: The Art, the Opportunities, and the Challenges.

The advent of nanotechnology has brought along new materials, techniques, and concepts, readily adaptable to lipid membrane-based biosensing. The transition from micro-sensors to nano-sensors is neither straightforward nor effortless, yet it leads to devices with superior analytical characteristics: ultra-low detectability, small sample volumes, better capabilities for integration, and more available bioelements and processes. Environmental monitoring remains a complicated field dealing with a large variety of pollutants, several decomposition products, or secondary chemicals produced ad hoc in the short- or medium term, many sub-systems affected variously, and many processes largely unknown. The new generation of lipid membranes, i.e., nanosensors, has the potential for developing monitors with site-specific analytical performance and operational stability, as well as analyte-tailored types of responses. This review presents the state-of-the art, the opportunities for niche applicability, and the challenges that lie ahead.

Artificial Lipid Membranes: Past, Present, and Future.

The multifaceted role of biological membranes prompted early the development of artificial lipid-based models with a primary view of reconstituting the natural functions in vitro so as to study and exploit chemoreception for sensor engineering. Over the years, a fair amount of knowledge on the artificial lipid membranes, as both, suspended or supported lipid films and liposomes, has been disseminated and has helped to diversify and expand initial scopes. Artificial lipid membranes can be constructed by several methods, stabilized by various means, functionalized in a variety of ways, experimented upon intensively, and broadly utilized in sensor development, drug testing, drug discovery or as molecular tools and research probes for elucidating the mechanics and the mechanisms of biological membranes. This paper reviews the state-of-the-art, discusses the diversity of applications, and presents future perspectives. The newly-introduced field of artificial cells further broadens the applicability of artificial membranes in studying the evolution of life.

Protein-Based Graphene Biosensors: Optimizing Artificial Chemoreception in Bilayer Lipid Membranes.

Proteinaceous moieties are critical elements in most detection systems, including biosensing platforms. Their potential is undoubtedly vast, yet many issues regarding their full exploitation remain unsolved. On the other hand, the biosensor formats with the higher marketability probabilities are enzyme in nature and electrochemical in concept. To no surprise, alternative materials for hosting catalysis within an electrode casing have received much attention lately to demonstrate a catalysis-coated device. Graphene and ZnO are presented as ideal materials to modify electrodes and biosensor platforms, especially in protein-based detection. Our group developed electrochemical sensors based on these nanomaterials for the sensitive detection of cholesterol using cholesterol oxidase incorporated in stabilized lipid films. A comparison between the two platforms is provided and discussed. In a broader sense, the not-so-remote prospect of quickly assembling a protein-based flexible biosensing detector to fulfill site-specific requirements is appealing to both university researchers and industry developers.

Construction of a simple optical sensor based on air stable lipid film with incorporated urease for the rapid detection of urea in milk.

This work describes the construction of a simple optical sensor for the rapid, selective and sensitive detection of urea in milk using air stable lipid films with incorporated urease. The lipid film is stabilized on a glass filter by polymerization using UV (ultra-violet) radiation prior its use. Methacrylic acid was the functional monomer, ethylene glycol dimethacrylate was the crosslinker and 2,2'-azobis-(2-methylpropionitrile) was the initiator. Urease is incorporated within this mixture prior to the polymerization. The presence of the enzyme in these films quenched this fluorescence and the colour became similar to that of the filters without the lipid films. A drop of aqueous solution of urea provided a "switching on" of the fluorescence which allows the rapid detection of this compound at the levels of 10(-8) M concentrations. The investigation of the effect of potent interferences included a wide range of compounds usually found in foods and also of proteins and lipids. These lipid membranes were used for the rapid detection of urea in milk.

Development of an electrochemical biosensor for the rapid detection of naphthalene acetic acid in fruits by using air stable lipid films with incorporated auxin-binding protein 1 receptor.

This work describes the investigations of electrochemical interactions of naphthalene acetic acid (NAA) with stabilized lipid films supported on a methacrylate polymer on a glass fiber filter with incorporated auxin-binding protein 1 receptor for the development of a biosensor for the rapid detection of this compound in fruits. NAA was injected into the flowing streams of a carrier electrolyte solution, the flow of the electrolyte solution stops and an ion current transient was obtained; the magnitude of the signal was correlated to NAA concentration, which could be determined at the micromolar level. NAA preconcentrates at the lipid membrane surface which causes dynamic alterations of the electrostatic fields and phase structure of membranes. The response times were ca. 5 min and naphthalene acetic acid was determined at concentration levels of microM. The effect of potent interferences included a wide range of compounds. The results showed no interferences from these compounds in concentration levels usually found in real samples. The method was applied for the determination of NAA in fruits and the reproducibility of the method was checked in about 100 samples. A quantitative method for the detection of NAA in fruits that can be complimentary to HPLC methods is provided in the present paper. These lipid films can be used as portable sensors for the rapid detection of NAA in fruits by non-skilled personnel.

Preparation of a selective receptor for carbofuran for the development of a simple optical spot test for its rapid detection using stabilized in air lipid films with incorporated receptor.

The present technique describes the preparation of a selective receptor for carbofuran and the development of a simple sensitive spot optical test for the rapid one-shot detection of carbofuran using stabilized lipid films supported on a methacrylate polymer on a glass fiber filter with incorporated artificial receptor. The selective receptor was synthesized by a chemical reaction using a resorcin[4]arene receptor by transforming all the -OH groups into phosphoryl groups. The lipid films without this receptor provided fluorescence under a UV lamp. The use of the receptor in these films quenched this fluorescence and the colour became similar to that of the filters without the lipid films. A drop of aqueous solution of carbofuran provided a "switching on" of the fluorescence which allows the rapid detection of this insecticide at the levels of 10(-9)M concentrations. The effect of potent interferences included a wide range of compounds. The results showed no interferences from these compounds in concentration levels usually found in real samples. The effect of interference of proteins and lipids was also examined. The reproducibility of the method was checked in about 100 samples and all of them were found to provide similar results. The device was tested/evaluated in real samples of fruits, vegetables and dairy products. Note that the colours of the filters remain stable for periods of more than 2 months.

Preparation of a selective receptor for ephedrine for the development of an optical spot test for the detection of ephedrine in human urine using stabilized in air lipid films with incorporated receptor.

The present technique describes the preparation of a selective receptor for ephedrine and a simple sensitive spot optical test for the rapid one-shot detection of ephedrine in human urine using lipid films with an incorporated receptor that are synthesized by a chemical reaction with a methacrylate polymer on a glass fiber filter. The selective receptor was synthesized using a resorcin[4]arene receptor and by transforming all the -OH groups into methoxy groups. The lipid films without this receptor provided fluorescence under a UV lamp. The use of the receptor in these films quenched this fluorescence, and the color became similar to that of the filters without the lipid films. A drop of aqueous solution of ephedrine provided a "switching on" of the fluorescence, which allows the rapid detection of this stimulant in human urine at the levels of 10(-8) M concentrations. The effect of potent interferences (i.e., proteins, lipids, ascorbic aid, glucose, leucine, glycine, tartrate, citrate, bicarbonate, and caffeine) was examined. The results showed no interferences from these compounds in concentration levels usually found in human urine samples. Dopamine was also investigated as a potent interfering agent, and the results have shown that the transformation of the hydroxy to methoxy groups has altered the selectivity of the receptor. This species does not cause interference at concentration levels lower than 10(-6) M. A drop of urine containing ephedrine provided also a "switching on" of the fluorescence, which allows the rapid detection of this stimulant in human urine at the levels of 10(-8) M concentrations. The reproducibility of the method was checked in approximately 100 samples, and all of them were found to provide similar results. Note that the colors of the filters remain stable for periods of more than 2 months.

An optical spot test for the detection of dopamine in human urine using stabilized in air lipid films.

The present technique describes a simple, sensitive spot test for the rapid one-shot detection of dopamine in human urine using lipid films with incorporated resorcin[4]arene receptor that are synthesized by a chemical reaction with a methacrylate polymer on a glass fiber filter. The lipid films without the receptor provided fluorescence under a UV lamp. The use of the receptor in these films quenched this fluorescence, and the color became similar to that of the filters without the lipid films. A drop of dopamine or urine containing this stimulant provided a "switching on" of the fluorescence, which allows the rapid detection of this stimulant in human urine at 10(-8) M concentrations. The novelty of the present work is that it opens new routes in the field of biosensing, i.e., development of sensitive, rapid, and simple methods for detecting species based on the fluorescence of the lipid membranes on a polymer film, and provides a spot test technique for the rapid detection of dopamine. The effect of potent interferences including a wide range of compounds usually found in human urine (i.e., ascorbic aid, glucose, leucine, glycine, tartrate, citrate, bicarbonate, and caffeine) was examined using an aqueous buffered solution that contained the potent interference and dopamine at two lower concentration levels (i.e., 3 x 10(-8)-10(-8) M). The effect of proteins and lipids was also investigated at these two lower dopamine concentration levels in aqueous buffered solution. The results showed no interferences from all these constituents at concentrations usually found in human urine samples; for example, albumin up to 3.22 g/L concentration levels did not provide any interference (i.e., no fluorescence). A drop of urine containing this stimulant provided similar results, i.e., a "switching on" of the fluorescence that allows a technique for the rapid detection of this stimulant in human urine at 10(-8) M concentrations. The technique is not based on a calibration graph but is a semiquantitative method for the detection of dopamine in real samples of urine that can be complimentary to HPLC methods. The difference in color between the samples containing dopamine at concentration levels of 10(-8)-10(-7) M can be easily distinguished by naked eye and a digital camera. An increase of dopamine concentration from 10(-8) to 10(-7) M makes the color more blue whereas the color of the filters remains purple in the blank test (i.e., addition of a urine sample without dopamine or dopamine at concentration levels of 10(-9) M to the filters that contain the lipid membranes with incorporated receptor). The reproducibility of the method was checked in approximately 100 samples, and all of them were found to provide similar results. Note that it was also found that the colors remain stable in the samples containing dopamine for periods of more than two months.

Biosensor for dopamine based on stabilized lipid films with incorporated resorcin[4]arene receptor.

This work reports a technique for the stabilization after storage in air of a lipid film with incorporated resorcin[4]arene receptor based biosensor for dopamine. Microporous filters composed of glass fibers (nominal pore sizes, 0.7 and 1.0 microm) were used as supports for the formation and stabilization of these devices and the lipid film is formed on the filter by polymerization prior its use. Methacrylic acid was the functional monomer, ethylene glycol dimethacrylate was the crosslinker and 2,2'-azobis-(2-methylpropionitrile) was the initiator. The stability of the lipid films by incorporation of a receptor for the preparation of stabilized lipid film biosensor is studied throughout this work. The response towards dopamine of the present stabilized for repetitive uses lipid membrane biosensor composed of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidic acid was compared with planar freely suspended bilayer lipid membranes (BLMs). The stabilized lipid membranes provided similar artificial ion gating events as BLMs in the form of transient signals and can function for repetitive uses after storage in air. However, the response of the stabilized lipid films was slower than that of the freely suspended BLMs. This will allow the practical use of the techniques for chemical sensing based on lipid films and commercialization of these devices, because it is now possible to prepare stabilized lipid film based biosensors and store them in the air.

A minisensor for the rapid screening of atenolol in pharmaceutical preparations based on surface-stabilized bilayer lipid membranes with incorporated DNA.

This work describes an electrochemical technique that is suitable for the rapid and sensitive screening of atenolol based on surface-stabilized bilayer lipid membranes (s-BLMs) composed from egg phosphatidylcholine (PC). The interactions of atenolol with s-BLMs produced electrochemical ion current increases that reproducible appeared within a few seconds after the exposure of the membranes to the drug. The current signal increase was related to the concentration of atenolol in bulk solution in the micromolar range. The present lipid film-based sensor provided fast response (i.e. on the order of a few seconds) to alterations of atenolol concentration (20 to 200 micro M) in electrolyte solution. ssDNA incorporated into s-BLMs can interact with atenolol, and decreased the detection limit of this drug by one order of magnitude. The oligomers used were single stranded deoxyribonucleic acids: thymidylic acid icosanucleotide terminated with a C-16 alkyl chain to assist incorporation into s-BLMs (5'-hexadecyl-deoxythymidylic acid icosanucleotide, dT(20)-C(16)). The electrochemical transduction of the interactions of atenolol with s-BLMs was applied in the determination of these compounds in pharmaceutical preparations by using the present minisensor.

Stabilized lipid film based biosensor for atenolol.

This work reports a technique for the stabilization after storage in air of a lipid film based biosensor for atenolol. Microporous filters composed of glass fibers (nominal pore sizes 0.7 and 1.0 microm) were used as supports for the formation and stabilization of these devices. The lipid film is formed on the filter by polymerization prior to its use. Methacrylic acid was the functional monomer, ethylene glycol dimethacrylate was the crosslinker and 2,2'-azobis-(2-methylpropionitrile) was the initiator. The method for preparation of stabilized lipid film biosensor is studied throughout this work. The response towards atenolol of these stabilized lipid membrane biosensor, for repetitive use, composed of phosphatidylcholine was compared with planar freely suspended bilayer lipid membranes (BLMs). The stabilized lipid membranes provided similar artificial ion gating events as BLMs in the form of transient signals and can function for repetitive uses after storage in air. This will allow the practical use of the techniques for chemical sensing based on lipid films and commercialization of these devices.

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.