Hybrid 2D-nanomaterials-based electrochemical immunosensing strategies for clinical biomarkers determination.

Owing to the outstanding conductivity and biocompatibility as well as numerous other fascinating properties of two-dimensional (2D)-nanomaterials, 2D-based nanohybrids have shown unparalleled superiorities in the field of electrochemical biosensors. This review highlights latest advances in electrochemical immunosensors for clinical biomarkers based on different hybrid 2D-nanomaterials. Particular attention will be given to hybrid nanostructures involving graphene and other graphene-like 2D-layered nanomaterials (GLNs). Several recent strategies for using such 2D-nanomaterial heterostructures in the development of modern immunosensors, both for tagging or modifying electrode transducers, are summarized and discussed. These hybrid nanocomposites, quite superior than their rival materials, will undoubtedly have an important impact within the near future and not only in clinical areas. Current challenges and future perspectives in this rapidly growing field are also outlined.

Study of calix[4]resorcinarene-dopamine complexation in mixed phospholipid monolayers formed at the air-water interface.

We have studied the physical properties of monolayers formed by calix[4]resorcinarene and in mixtures with dipalmitoyl phosphatidylcholine (DPPC) in various molar ratios formed at the air-water interface and at presence of dopamine in water subphase by means of measurements of surface pressure and dipole potential. We showed that both calix[4]resorcinarene as well as its mixture with DPPC form stable monolayers at the water subphase. The presence of dopamine resulted in an increase of the mean molecular area and in a decrease of the compressibility modulus of the monolayers. For mixed monolayers at higher content of calix[4]resorcinarene (> 0.2 molar fraction) a deviation from ideal miscibility took place especially for monolayers in a solid state. This can be connected with formation of aggregates of calix[4] resorcinarene. Lowest miscibility and weakest interaction of dopamine with a monolayer was observed for calix[4]resorcinarene molar fraction of 0.33 in the monolayer.

Liposome aggregation in presence of the sweeteners cyclamate and saccharine.

The interaction of the sweeteners saccharine and cyclamate with large unilamellar liposomes and planar bilayer lipid membranes (BLM) was studied. Application of the methods of light scattering and sound velocimetry showed that saccharine induces aggregation of liposomes, while cyclamate probably caused increase of the hydration of liposome surface. The sweeteners induced changes of BLM compressibility in a direction perpendicular to the membrane plane. The cyclamate induced considerably larger decreases in the elasticity module than saccharine. The obtained results show that both saccharine and cyclamate interacts with the surface of lipid bilayer and could modify the physical properties of lipid membranes.

Selective continuous monitoring and analysis of mixtures of acesulfame-K, cyclamate, and saccharin in artificial sweetener tablets, diet soft drinks, yogurts, and wines using filter-supported bilayer lipid membranes.

This work describes a technique for the rapid and sensitive electrochemical flow injection monitoring and analysis of mixtures of the artificial sweeteners acesulfame-K, cyclamate, and saccharin using stabilized systems of filter-supported bilayer lipid membranes (BLMs). Injections of artificial sweeteners were made into flowing streams of a carrier electrolyte solution, and a transient current signal with duration of seconds reproducibly appeared in less than < 1 min after exposure of the lipid membranes to the artificial sweeteners. The magnitude of this signal was linearly related to the concentration of artificial sweeteners, which could be determined at micromolar levels. Repetitive cycles of injection of artificial sweeteners have shown no signal degradation during each cycle (30 sequential injections). The time of appearance of the transient response was different for each artificial sweetener and increased in the order of cyclamic acid, acesulfame-K, and saccharin. The difference in time of response has allowed selective detection and analysis of these artificial sweeteners in mixtures. The effect of potent interferences, including a wide range of compounds usually found in foods, proteins, and lipids was investigated. The results showed no interferences from these constituents of real food samples. The major interference from proteins (most common in lipid-film-based biosensors) can be eliminated by modulation of the carrier solution that does not allow adsorption of these compounds in BLMs. The technique was applied in real food samples, that is, in artificial sweetener tablets, diet soft drinks, wines, and yogurts that contain mixtures of these artificial sweeteners with aspartame and other compounds. A comparison of results using the present method and that of an Official Method of Analysis showed good agreement between the two methods.

Flow injection monitoring and analysis of mixtures of hydrazine compounds using filter-supported bilayer lipid membranes with incorporated DNA.

This work describes a technique for the rapid and sensitive electrochemical flow injection monitoring and analysis of mixtures of hydrazine compounds using stabilized systems of filter-supported bilayer lipid membranes (BLMs) composed of egg phosphatidylcholine (egg PC) with incorporated DNA. Injections of hydrazines were made into flowing streams of a carrier electrolyte solution, and a transient current signal with a duration of seconds reproducibly appeared in less than one min after exposure of the DNA-modified lipid membranes to the hydrazines. The magnitude of this signal was linearly related to the concentration of hydrazines, which could be determined at sub-micromolar levels. Repetitive cycles of injection of hydrazines have shown no signal degradation during each cycle (30 sequential injections). The time of appearance of the transient response was different for each hydrazine and increased in the order of hydrazine, methylhydrazine or dimethylhydrazine, and phenylhydrazine. The difference in time of response has allowed selective detection and analysis of these hydrazines in mixtures.

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.

Binding of avidin modified antibody to biotinylated metal supported membranes and liposomes changes the physical properties of lipid bilayer.

Various methods have been applied to study the physical properties of metal supported bilayer lipid membranes (s-BLM) and large unilamellar liposomes containing biotinylated phospholipids during the binding of IgG modified by avidin. Electrostriction method applied to s-BLM showed that addition of avidin-IgG (A-IgG) complex in a small concentration (0.2 mumol/l) resulted in an approximately twofold decrease of membrane capacitance, C, increase of elasticity modulus in direction perpendicular to the membrane plane, E perpendicular, by 5-20%, increase in intrinsic membrane potential, delta phi m, by approximately 30 mV, and an approximately 5-15% increase of the coefficient of dynamic viscosity, eta. The method of ultrasonic velocimetry showed that addition into the suspension of liposomes of unmodified IgG did not induce any changes in ultrasound velocity, while addition of A-IgG complex in a concentration range of 0-1 mumol/l led to an increase of the velocity number [u]. The plot of changes of [u] versus A-IgG concentration has a tendency to saturate at concentrations above 0.5 mumol/l, which suggests long-range interactions in the membrane induced by strong binding of A-IgG to the biotin sites on the membrane. Probably, this binding leads to a decrease of the coefficient of adiabatic compressibility of liposomes.

Flow injection monitoring of aflatoxin M1 in milk and milk preparations using filter-supported bilayer lipid membranes.

This work describes a technique for the rapid and sensitive electrochemical flow injection monitoring of aflatoxin M1 (AFM1) using stabilized systems of filter-supported bilayer lipid membranes (BLMs). Injections of AFM1 were made into flowing streams of a carrier electrolyte solution, and a transient current signal with a duration of seconds reproducibly appeared less than 10 s after exposure of the lipid membranes to the toxin. The magnitude of this signal was linearly related to the concentration of AFM1, with detection limits at the subnanomolar level. The mechanism of signal generation was investigated by differential scanning calorimetric experiments. The technique was applied for the rapid flow injection determination of AFM1 in milk and milk preparations. The effect of potent interferences such as proteins and lipids was investigated, and the results show that interferences from these milk constituents can be eliminated by modulation of the flow rate of the carrier solution so as not to allow adsorption of these compounds in BLMs. AFM1 could be determined in continuous flowing systems with a rate of at least 4 samples min-1. Repetitive cycles of injection of AFM1 showed no signal degradation during each cycle.

Stabilized filter-supported bilayer lipid membranes (BLMs) for automated flow monitoring of compounds of clinical, pharmaceutical, environmental and industrial interest.

This paper describes the results of analytical applications of electrochemical biosensors based on bilayer lipid membranes (BLMs) for the automated rapid and sensitive flow monitoring of substrates of hydrolytic enzymes, antigens and triazine herbicides. BLMs, composed of mixtures of egg phosphatidylcholine (egg PC) and dipalmitoylphosphatidic acid (DPPA), were supported on ultrafiltration membranes (glass microfibre or polycarbonate filters) which were found to enhance their stability for flow experiments. The proteins (enzymes, antibodies) were incorporated into a floating lipid matrix at an air-electrolyte interface, and then a casting procedure was used to deliver the lipid onto the filter supports for BLM formation. Injections of the analyte were made into flowing streams of the carrier electrolyte solution and a current transient signal was obtained with a magnitude related to the analyte concentration. Substrates of hydrolytic enzyme reactions (acetylcholine, urea and penicillin) could be determined at the micromolar level with a maximum rate of 220 samples/h, whereas antigens (thyroxin) and triazine herbicides (simazine, atrazine and propazine) could be monitored at the nanomolar level in less than 2 min. The time of appearance of the transient response obtained for herbicides was increased to the order of simazine, atrazine and propazine which has permitted analysis of these triazines in mixtures.

A triazine herbicide minisensor based on surface-stabilized bilayer lipid membranes.

This work describes an electrochemical technique that is suitable for rapid and sensitive screening of the triazine herbicides simazine, atrazine, and propazine. Egg phosphatidylcholine and dipalmitoylphosphatidic acid (DPPA) were used for the formation of self-assembled bilayer lipid membranes supported on silver wire (s-BLMs). Evidence that BLMs could form on silver wires was collected by means of ellipsometry which was done to investigate samples consisting of lipids deposited on planar reflective silver films. The interactions of triazines with s-BLMs produced electrochemical ion current increases which reproducibly appeared within ∼10 s after exposure of the lipid membranes to the herbicides. The sensitivity of the response was maximized by use of BLMs composed of 35% (w/w) DPPA and by alteration of the phase distribution within membranes by the introduction of 1.0 mM calcium ions in bulk solution. The mechanism of signal generation could be a result of rapid adsorption of the triazine on the surface of s-BLMs with a consequent rapid reorganization of the electrostatics of the membrane. The magnitude of the current signal was linearly related to the herbicide concentration, which could be determined at the nanomolar level. The present triazine minisensor exhibited good mechanical stability and longevity (routinely over 48 h), reproducible response characteristics (i.e., sensitivity and response to a given concentration of triazine in solution), fast response times, and low detection limits. The sensor can be simply and reliably fabricated at low cost. Studies have shown high selectivity for triazines in the presence of insecticides and pesticides.

Fourier analysis of potential oscillations of a liquid membrane for the discrimination of taste substances.

Electrical potential oscillations were obtained across a liquid membrane composed of nitrobenzene/picric acid placed between two aqueous phases in the presence of various taste (i.e. salty, sweet and bitter) substances. The influence of these compounds on electrical oscillations was studied using Fourier analysis to establish a "fingerprint" of the substance that can be correlated with its taste index. Various concentrations of each substance were tested to obtain a Fourier spectrum with discrete peaks which can be further processed. The electrical oscillations consisted of a number of weak damped oscillators, and the Fourier spectra of these signals were found to have a number of discrete peaks of decreasing amplitude at low frequencies (0-0.5 Hz). A correlation of the frequency of the first peak of the Fourier spectrum with the taste index was found for bitter substances, whereas for salty substances the amplitude of the first two peaks of the spectrum was correlated with the taste index.

Evaluation of a glassy carbon electrode modified by a bilayer lipid membrane with incorporated DNA.

The objective of the present work was the evaluation and characterization of a glassy carbon (GC) electrode modified by a bilayer lipid membrane (BLM) with incorporated single-stranded deoxyribonucleic acid fss DNA). Various procedures were developed and tested for the incorporation of ss DNA at the electrode modified by the lipidic membrane: Differential pulse voltammetry (i.e. oxidation of guanine and adenine residues) was used to monitor the incorporation of ss DNA at the GC electrode modified by the BLM. The results have shown that the lipid membrane enhances the stability of ss DNA during a "medium-exchange" of the electrode and prohibits its diffusion from the electrode surface. The third scheme was proven to be the most appropriate as both electrode modification by the BLM and DNA adsorption occur in one stage and much faster (as no BLM thinning process is required) as compared to the former two techniques; furthermore, maximized loading of DNA in BLMs is achieved which reduces by ca. 10-fold the DNA amounts that can be detected electrochemically. Conventional planar "free-suspended" and self-assembled metal supported BLMs were used to monitor in situ the incorporation of ss DNA in these membranes. The results have shown that the adsorption of ss DNA at lipid membranes (as a medium for DNA incorporation on an electrode surface) can occur much faster, using milder conditions and smaller amounts of DNA than by previously described techniques.

Ammonium ion minisensors form self-assembled bilayer lipid membranes using gramicidin as an ionophore. Modulation of ammonium selectivity by platelet-activating factor.

The present paper reports the electrochemical investigation of ion transport through self-assembled bilayer lipid membranes on a metal support and represents a technology suitable for development of an ammonium ion minisensor. Gramicidin D was used as a channel-forming ionophore for selective conduction of ammonium ion through lipid bilayers composed of egg phosphatidylycholine. The ammonium ion sensor exhibited good mechanical stability and longevity (routinely over 48 h) and constant sensitivity and response to a given concentration of ammonium ion in solution. The use of stabilized metal-supported BLMs has allowed the electrochemical investigation of the reversibility of response to ammonium ions and of ionophore binding to lipid membranes. The effects of pH and some possible interferents were examined. Semisynthetic platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, AGEPC) was found to enhance transport of ammonium ions by gramicidin and to reduce potassium interference, whereas the non-acetylated derivative of AGEPC did not exert any alterations in transport ammonium ions. The present microfabricated ammonium sensor based on thin lipid film technology provides advantages of extremely fast response times (to millisecond speeds) to alterations of ammonium ion concentration (0.02-5 mmol L-1), high selectivity to ammonium ions in the presence of volatile amines, and the capability of analyzing small volumes of samples. A detection limit of ammonium ions of approximately 1 x 10(-6) M was attained using BLMs modified with PAF. Furthermore, a device can now simply and reliably be fabricated at low cost and therefore can be used as a disposable sensor.

Bilayer lipid membranes for flow injection monitoring of acetylcholine, urea, and penicillin.

This work describes a technique for the rapid and sensitive determination of acetylcholine, urea, and penicillin in flowing solution streams using stabilized systems of solventless bilayer lipid membranes (BLMs). This method of monitoring substrates of hydrolytic enzyme reactions made use of BLMs which were supported on ultrafiltration membranes such as polycarbonate and glass microfiber; these filter membranes were found to enhance the stability of BLMs for uses in flow injection experiments. The enzymes were immobilized on BLMs by incorporating the protein solution into the lipid matrix at the air/electrolyte interface before the BLM formation, followed by injections of the substrates into flowing streams of a carrier electrolyte solution. Hydronium ions produced by the enzymatic reaction at the BLM surface caused dynamic alterations of the electrostatic fields and phase structure of BLMs, and as a result ion current transients were obtained; the magnitude of these signals was correlated to the substrate concentration, which could be determined at the micromolar level. The response times were ca. 10 s, and acetylcholine, urea, and penicillin could be determined in continuous flowing systems with a maximum rate of 220 samples/h. It is expected that this analytical utility of stabilized BLMs for flow stream uses will provide new opportunities in this strategy of chemical sensing.

The bilayer lipid membrane as a generic electrochemical transducer of hydrolytic enzyme reactions.

Bilayer lipid membranes (BLMs) can be used as generic transducers to monitor hydrolytic enzyme reactions occurring at the membrane surface. The representative enzymatic reactions presented herein were between membrane associated urease and penicillinase with urea and penicillin, respectively. Transient electrochemical signals from BLMs which contained enzyme were obtained by proper selection of the lipid composition of membranes. Negatively charged lipid membranes composed of egg phosphatidylcholine (PC) and 35% dipalmitoylphosphatidic acid were used for this purpose. The results were consistent with an electrostatic mechanism of perturbation of the surface structure of the BLMs, where changes of local hydronium ion activity associated with the enzymatic reaction altered the extent of ionization of the headgroups of the acidic constituent of the membranes, 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 microM to mM levels. The results indicate that BLMs can be used as generic selective electrochemical transducers and as switchable biosensors to monitor rapid enzymatic reactions which alter pH.

Reliable and facile method for preparation of solventless bilayer lipid membranes for electroanalytical investigations.

Bilayer lipid membranes continue to be of interest as elements for development and investigation of chemically-modified electrodes and biosensors, yet also continue to be difficult to prepare and replicate with precision. A simplified and reliable technique for the rapid formation of solventless bilayer lipid membranes is described, and the method has been shown to produce membranes which nominally vary by only 5-10% with respect to ion conductivity. Methods for rapid physical and chemical characterization of these membranes for establishment of reproducibility and quality are given.

Ion permeability through bilayer lipid membranes for biosensor development: control by chemical modification of interfacial regions between phase domains.

Based on the results of studies on cystic fibrosis, which implicated hydroxystearic acid (HSA) as a contributing factor in altered biomembrane function, solvent-free bilayer lipid membranes (BLMs) and monolayer films were prepared from a lipid mixture containing (by mass) 34% phosphatidylcholine, 19% dipalmitoylphosphatidyl serine, 47% cholesterol and variable amounts of 10- and 12-HSA (0-50%). Ion currents, resulting from K+ permeation through BLMs that were supported in 0.1 mol dm-3 KCl solutions buffered to pH 7.4, were monitored with use of a d.c. circuit. The structures of monolayer films at the air-water interface of a Langmuir-Blodgett trough were studied by pressure-area correlations and by further correlation with microscopic phase separation as revealed by fluorescence microscopy. In order to elucidate the role of the hydroxyl moieties in ion permeability, the transmembrane ion current was corrected for the effect of the negative surface charge of the carboxylic acid by replacement of the HSA component with stearic acid. The ion current was found to increase with the molar proportion of the HSAs. Two models for ion conduction through BLMs were considered: 'hopping' via hydrophilic sites within the hydrophobic zone of the BLMs, introduced by the hydroxyl moiety of 10- or 12-HSA; and transport through interfacial regions between phase domains that represent areas of low steric density and low structural order within monolayers. Although the two mechanisms are not distinct, the ion permeability results indicate a change in the response of ion current to HSA concentration at 35 mol-%, suggesting a change in the relative proportion of the mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamic response characteristics of the potentiometric carbon dioxide sensor for the determination of aspartame.

The dynamic response characteristics of a carbon dioxide gas sensor were studied to determine the potential for application of the device to the kinetic assay of substrate(s) under pseudo first-order kinetics. The dependence of the time constant on the concentration of carbon dioxide was determined by using convolution mathematics to analyse potentiometric changes caused by abrupt alterations of gas concentration. The operational conditions of the CO2 sensor were optimised for the development of enzyme electrodes, so that the mass-transport phenomena occurring during the course of the enzymic reactions were enhanced. As a result, the kinetic analysis of substrate(s) was performed more rapidly (2-6 min), with greater sensitivity and with an improved detection limit (10-5 M). A kinetic reaction-rate method for the determination of aspartame in dietary foodstuffs is proposed as a rapid and inexpensive alternative to a classical high-performance liquid chromatographic method.

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%.