Recognition of 6-benzyladenine using a molecularly imprinted membrane on a cellulose acetate support

A molecularly imprinted polymer membrane was prepared on a cellulose acetate support by the photopolymerization of methacrylic acid and a cross linker, ethyleneglycol dimethacrylate, in the presence of the template molecules of 6-benzyladenine (BA). The polymeric membrane morphologies were visualized by scanning electron microscopy and its selectivity was evaluated by a permeation test. The association ratio and apparent association constant of the complex formed between the methacrylic acid and BA were determined by cyclic voltammetry, and are 1 : 1 and 204.9, respectively. These results indicated that there existed some complementary cavities on the imprinted membrane corresponding in size, shape, and functional groups to the template molecules of BA. Hence, the imprinted membrane was able to recognize BA. It is predicted that this molecularly imprinted membrane may be applicable to the assay of BA or for the preparation of a molecularly imprinted polymer sensor for the determination of BA in plant samples. The text was submitted by the authors in English.     

Urinary l‐kynurenine quantification and selective extraction through a molecularly imprinted solid‐phase extraction device

l ‐Kynurenine is an endogenous metabolite generated by the catabolic pathway of l ‐tryptophan and it could be a potential biomarker to test the efficacy of several checkpoint inhibitors that have already reached the clinical trials in the antitumor therapy. Thus, a molecularly imprinted polymer specific for the recognition of this metabolite was synthesized and used as innovative system in solid‐phase extraction technique for the specific extraction and quantification of l ‐kynurenine in human urine. The off‐line system was firstly tested on l ‐kynurenine standard solutions, allowing recoveries up to 97.7% (relative standard deviation = 2.2%) and then applied to fortified and deproteinated human urine samples, where a recovery of 84.1% (relative standard deviation = 3.1%) was obtained. The method was validated and it revealed a good linearity in the range of 0.157–20 μg/mL (r² = 0.9992). The optimized procedure demonstrated a good feasibility on biological samples, allowing a ready quantification of l ‐kynurenine in the human urine, where the metabolite was found at a very low concentration (0.80 μg/mL). The extraction system developed could attract attention of pharmaceutical industries for l ‐kynurenine production as potential drug in the treatment of autoimmune disorders through its extraction and purification from biological matrixes.     

A reservoir-type controlled release device using aqueous-organic partitioning and a porous membrane

A reservoir-type controlled release device based on aqueous-organic partitioning is described. The reservoir is bounded by a microporous or porous membrane, either a hollow fiber or a flat film. The agent partitions between phases at the aqueous-organic interface of the reservoir and the pore mouth, and then diffuses through the membrane pore into a surrounding aqueous solution. The partition coefficient significantly influences the rate of release of the agent. The performance of the system is evaluated using model agents. Controlled release from a reservoir containing a pure organic liquid agent is demonstrated using toluene. Zero-order release is achieved for benzoic acid partitioning from an organic reservoir into water-filled pores, and for nicotine partitioning from an aqueous reservoir into organic-filled pores. Studies using benzoic acid demonstrate the effectiveness of a thin, nonporous coating on slowing the rate of release. A fast-dissolving suspension of benzoic acid in decanol extends the duration of zero-order release. Two agents, nicotine and caffeine, are released simultaneously and independently from a divided reservoir. A simplified mathematical model is presented, and experimental results compared well with those predicted by the model.     

Detection of parathion methyl using a surface plasmon resonance sensor combined with molecularly imprinted films

An ultra-sensitive and highly selective parathion methyl(PM) detection method by surface plasmon resonance(SPR) combined with molecularly imprinted films(MIF) was developed. The PM-imprinted film was prepared by thermo initiated polymerization on the bare Au surface of an SPR sensor chip.Template PM molecules were quickly removed by an organic solution of acetonitrile/acetic acid(9:1,v/v), causing a shift of 0.58 in SPR angle. In the concentrations range of 10à13–10à10mol/L, the refractive index showed a gradual increase with higher concentrations of template PM and the changes of SPR angles were linear with the negative logarithm of PM concentrations. In the experiment, the minimum detectable concentration was 10à13mol/L. The selectivity of the thin PM-imprinted film against diuron,tetrachlorvinphose and fenitrothion was examined, but no observable binding was detected. The results in the experiment suggested that the MIF had the advantages of high sensitivity and selectivity.     

Detection and quantification of leucyl aminopeptidase after native electrophoresis using leucine-p-nitroanilide

A general method for detecting leucyl aminopeptidase activity after native polyacrylamide gel electrophoresis (PAGE) in situ is described. The method is based on diazotization of p-nitroaniline, liberated in the polyacrylamide gel by leucyl aminopeptidase action on leucine-p-nitroanilide (LpNA) and subsequent coupling with a chromogen, 1-naphthylamine, until a pink azo dye product at the position of enzyme activity is obtained. A possible use of this technique for leucyl aminopeptidase detection and quantification is indicated. This method was found to be reproducible with the coefficient of variation below 15% for a 32-fold range, while the colored area of enzyme activity was in linear dependence to enzyme activity. Applications of this method with some other aminoacyl-p-nitroanilides and for detection of kidney bean leucyl aminopeptidase isoforms are demonstrated.     

Passive transport of C60 fullerenes through a lipid membrane: a molecular dynamics simulation study

To investigate the implications of the unique properties of fullerenes on their interaction with and passive transport into lipid membranes, atomistic molecular dynamics simulations of a C60 fullerene in a fully hydrated di-myristoyl-phoshatidylcholine lipid membrane have been carried out. In these simulations the free energy and the diffusivity of the fullerene were obtained as a function of its position within the membrane. These properties were utilized to calculate the permeability of fullerenes through the lipid membrane. Simulations reveal that the free energy decreases as the fullerene passes from the aqueous phase, through the head group layer and into the hydrophobic core of the membrane. This decrease in free energy is not due to hydrophobic interactions but rather to stronger van der Waals (dispersion) interactions between the fullerene and the membrane compared to those between the fullerene and (bulk) water. It was found that there is no free energy barrier for transport of a fullerene from the aqueous phase into the lipid core of the membrane. In combination with strong partitioning of the fullerenes into the lipidic core of the membrane, this "barrierless" penetration results in an astonishingly large permeability of fullerenes through the lipid membrane, greater than observed for any other known penetrant. When the strength of the dispersion interactions between the fullerene and its surroundings is reduced in the simulations, thereby emulating a nanometer sized hydrophobic particle, a large free energy barrier for penetration of the head group layer emerges, indicating that the large permeability of fullerenes through lipid membranes is a result of their unique interaction with their surrounding medium.     

Reversible redox of NADH and NAD+ at a hybrid lipid bilayer membrane using ubiquinone

Here, we report the reversible interconversion between NADH and NAD(+) at a low overpotential, which is in part mediated by ubiquinone embedded in a biomimetic membrane to mimic the initial stages of respiration. This system can be used as a platform to examine biologically relevant electroactive molecules embedded in a natural membrane environment and provide new insights into the mechanism of biological redox cycling.     

Detection of organophosphorus compounds with semiconductor gas sensors using adsorption preconcentration

Effective concentrators for organophosphorus compounds, highly sensitive semiconductor sensors, and a prototype of a detector for specific determination of low concentrations of organophosphorus compounds in air were developed. It is preferable to use as concentrating sorbents silicates with low density of surface hydroxy groups, ensuring high degree of recovery of the target compound by thermal desorption without its decomposition. SnO₂ modified with RuO₂ is the most sensitive sensor material.     

Detection of supported lipid bilayers using their electric charge

We describe an electronic detection method for charged lipid bilayers supported on a Si 3N 4/SiO 2/Si substrate. The flat-band voltage was used to monitor the charge of the bilayers. We show that the flat-band voltage varies with lipid adsorption depending on the polarity and mole ratio of the charged lipids, the salt concentration, and the surface coverage. Cationic and anionic bilayers produced a decrease and an increase in the flat-band voltage, respectively. The voltage change increased as the percentage of charged lipid components was elevated in the planar bilayers with full surface coverage. In addition, the voltage variation increased when the salt concentration was decreased or when the surface coverage of planar bilayer patches was increased. These results demonstrate that charged bilayers can be detected from the field effect that they exert on a solid support.     

Selective recognition of ovalbumin using a molecularly imprinted polymer

Micro-contact imprinting has been used to form thin-film molecular imprints of ovalbumin (OVA) in polymers supported on glass slides. Thermocalorimetric data was used to optimise the choice of functional monomer and cross-linker to maximise selectivity and minimise non-specific recognition.A polymer comprising polyethyleneglycol 400 dimethacrylate (95 vol.%) and methacrylic acid (5 vol.%) showed both maximum recognition for OVA when made as a molecularly imprinted polymer (MIP), and minimal recognition when made as a non-imprinted, i.e. control polymer. OVA rebinding to the molecularly imprinted polymer, from a buffered 2 µM OVA solution, was 1.55 × 10^{− 11} mol cm^{− 2}, while the control polymer showed 10-fold less re-binding, i.e. 0.154 × 10^{− 11} mol cm^{− 2}.Experiments in which human serum albumin (HSA), conalbumin, ovomucoid or lysozyme, were re-bound to the polymers, either as single proteins or in competition with OVA, showed them to have low affinity for the polymer formulation used. Of the competing proteins examined, in non-competitive binding experiments, HSA showed the greatest affinity 0.45 × 10^{− 11} mol cm^{− 2} for the OVA imprinted polymer. In two protein competition experiments, i.e. with OVA and a competing protein present at equal concentrations (2 µM), OVA binding to the OVA imprinted polymer was in all cases significantly greater than that of the competitor.     

Amperometric morphine sensing using a molecularly imprinted polymer-modified electrode

This study incorporates morphine into a molecularly imprinted polymer (MIP) for the amperometric detection of morphine. The polymer, poly(3,4-ethylenedioxythiophene), PEDOT, is an electroactive film that catalyzes morphine oxidation and lowers the oxidization potential on an indium tin oxide (ITO) electrode. The MIP-PEDOT modified electrode is prepared by electropolymerizing PEDOT onto an ITO electrode in a 0.1 M LiClO₄ solution with template addition (morphine). After template molecule extraction, the oxidizing current of the MIP-PEDOT modified electrode is measured in a 0.1 M KCl solution (pH = 5.3) at 0.75 V (versus Ag/AgCl/sat’d KCl) with the morphine concentration varying in the 0.1-5 mM range. A linear range, displaying the relationship between steady-state currents and morphine concentrations, from 0.1 to 1 mM, is obtained. The proposed amperometric sensor could be used for morphine detection with a sensitivity of 91.86 μA/cm² per mM. A detection limit of 0.2 mM at a signal-to-noise ratio of 3 is achieved. Moreover, the proposed method can discriminate between morphine and its analogs, such as codeine.     

Detection and relative quantification of proteins by surface enhanced Raman using isotopic labels

Accurate quantification of protein content and composition has been achieved using isotope-edited surface enhanced resonance Raman spectroscopy. Synthesis of isotopomeric Rhodamine dye-linked bioconjugation reagents enabled direct labeling of surface lysines on a variety of proteins. When separated in polyacrylamide gels and stained with silver nanoparticles. The spectral signatures reflect the expected statistical distribution of isotopomeric labels on the labeled proteins in the gel matrix format without interference from protein features.     

Probing the influence of cis-trans isomers on model lipid membrane fluidity using cis-parinaric acid and a stop-flow technique

Stop-flow experiments exploiting the fluorescence of cis-parinaric acid in monounsaturated lipid vesicles allow the model membrane behaviour, notably the membrane fluidity, to be correlated to the cis:trans lipid ratios.     

Electrochemical sensor for 2,4-dichlorophenoxy acetic acid using molecularly imprinted polypyrrole membrane as recognition element

An electrochemical sensor based on molecularly imprinted polypyrrole membranes is reported for the determination of 2,4-dichlorophenoxy acetic acid (2,4-D). The sensor was prepared by electropolymerization of pyrrole on a glassy carbon electrode in the presence of 2,4-D as a template. The template was removed by overoxidation at +1.3 V in buffer solution. The sensor can effectively improve the reductive properties of 2,4-D and eliminate interferences by other pesticides and electroactive species. The peak current at -0.78 V is linear with the concentration of 2,4-D from 1.0 to 10 µM, the detection limit is 0.83 µM (at 3σ), and the relative standard deviation is 3.9% (at 5.0 µM of 2,4-D; n?=?7). The method has been successfully applied to the determination of 2,4-D in environmental water samples, with recovery rates ranging from 92% to 108%.     

Detection of nitrogen dioxide using mixed tungsten oxide-based thick film semiconductor sensor

The thick film semiconductor sensor for NO₂ gas detection was fabricated by screen-printing method using a mixed WO₃-based as sensing material. The sensing characteristics, such as response time, response linearity, sensitivity, working range, cross sensitivity, and long-term stability were further studied by using a WO₃-based mixed with different metal oxides (SnO₂, TiO₂ and In₂O₃) and doped with noble metals (Au, Pd and Pt) as sensing materials was observed. The highest sensitivity for low concentrations (<16 mg l⁻¹) was observed using WO₃-based mixed with In₂O₃ or TiO₂. The NO₂ gas sensor showing the fastest response and recovery time (both within 2 min), good linearity (Y=0.606X+0.788 R²=0.991) for gas concentrations from 3 to 310 mg l⁻¹, low resistance (3 MΩ), high sensitivity, undesirable cross sensitivity effect and good long-term stability (at least 120 days) using WO₃-SnO₂-Au as sensing material.     

Measuring the partitioning kinetics of membrane biomolecules using patterned two-phase coexistant lipid bilayers

We report a new method for measuring the partitioning kinetics of membrane biomolecules to different lipid phases using a patterned supported lipid bilayer (SLB) platform composed of liquid-ordered (lipid raft) and liquid-disordered (unsaturated lipid-rich) coexistent phases. This new approach removes the challenges in measuring partitioning kinetics using current in vitro methods due to their lack of spatial and temporal control of where phase separation occurs and when target biomolecules meet those phases. The laminar flow configuration inside a microfluidic channel allows us to pattern SLBs with coexistent phases in predetermined locations and thus eliminates the need for additional components to label the phases. Using a hydrodynamic force provided by the bulk flow in the microchannel, target membrane-bound species to be assayed can be transported in the bilayers. The predefined location of stably coexistent phases, in addition to the controllable movement of the target species, allows us to control and monitor when and where the target molecules approach or leave different lipid phases. Using this approach with appropriate experimental designs, we obtain the association and dissociation kinetic parameters for three membrane-bound species, including the glycolipid G(M1), an important cell signaling molecule. We examine two different versions of G(M1) and conclude that structural differences between them impact the kinetics of association of these molecules to raft-like phases. We also discuss the possibilities and limitations for this method. One possible extension is measuring the partitioning kinetics of other glycolipids or lipid-linked proteins with posttranslational modifications to provide insight into how structural factors, membrane compositions, and environmental factors influence dynamic partitioning.     

Discrimination of peptides by using a molecularly imprinted piezoelectric biosensor

Based on the direct formation of a molecularly imprinted polymer on gold electrodes, we have developed a peptide sensor for the detection of low-molecular-weight peptides. A new cross-linking monomer, (N-Acr-L-Cys-NHBn)(2), was employed to attach the surface of the chip and to copolymerize with other monomers. Interestingly, N-benzylacrylamide participates in the polymerization and recognition is carried out in an aqueous environment. By using quartz crystal microbalance detection, short peptides can be monitored by their interaction with plastic antibodies specific for the target peptides. The selectivity of molecularly imprinted polymers and the sensitivity of such artificial biosensors have been combined to differentiate between traces of oxytocin and vasopressin to the ng mL(-1) scale.     

Single molecule measurements within individual membrane-bound ion channels using a polymer-based bilayer lipid membrane chip

The measurement of single poly(ethylene glycol) (PEG) molecules interacting with individual bilayer lipid membrane-bound ion channels is presented. Measurements were performed within a polymer microfluidic system including an open-well bilayer lipid membrane formation site, integrated Ag/AgCl reference electrodes for on-chip electrical measurements, and multiple microchannels for independent ion channel and analyte delivery. Details of chip fabrication, bilayer membrane formation, and alpha-hemolysin ion channel incorporation are discussed, and measurements of interactions between the membrane-bound ion channels and single PEG molecules are presented.     

Dynamic calibration and dissolved gas analysis using membrane inlet mass spectrometry for the quantification of cell respiration

A membrane inlet mass spectrometer connected to a miniaturized reactor was applied for dynamic dissolved gas analysis. Cell samples were taken from 7 mL shake flask cultures of Corynebacterium glutamicum ATCC 13032, and transferred to the 12 mL miniaturized reactor. There, oxygen uptake and carbon dioxide and its mass isotopomer production rates were determined using a new experimental procedure and applying nonlinear model equations. A novel dynamic method for the calibration of the membrane inlet mass spectrometer using first-order dynamics was developed. To derive total dissolved concentration of all carbon dioxide species (C(T)) from dissolved carbon dioxide concentration ([CO(2)](aq)), the ratio of C(T) to [CO(2)](aq) was determined by nonlinear parameter estimation, whereas the mass transfer coefficient of CO(2) was determined by the Wilke-Chang correlation. Subsequently, the suitability of the model equations for respiration measurements was examined using residual analysis and the Jarque-Bera hypothesis test. The resulting residuals were found to be random with normal distribution, which proved the adequacy of the application of the model for cell respiration analysis. Hence, dynamic changes in respiration activities could be accurately analyzed using membrane inlet mass spectrometry with the novel calibration method.