A biosensor based on the membrane protein lactose permease (original) (raw)
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Biosensors based on membrane transport proteins
Biosensors and Bioelectronics, 1991
We propose a novel class of biosensors based on membrane bound receptors or transport proteins as the sensing element The protein is incorporated in a planar lipid bilayer which covers the transducer. The transducer may detect an electric current, a voltage, or a change in fluorescence. A prototype lactose sensor is presented which consists of a quartz slide covered by a lipid membrane containing the protein lactose permease from Escherichia coli. This protein is a lactose/H+ cotransporter, hence lactose in the external medium initiates lactose/H+ cotransport across the lipid membrane. This leads to a rise in proton concentration in the small volume between the lipid membrane and the quartz surface which can be detected by a pH-sensitive fluorescence dye.
Protein Science, 1994
Lactose transport in membrane vesicles containing lactose permease with a single Cys residue in place of Val 315 is inactivated by N-ethylmaleimide in a manner that is stimulated by substrate or by a H + electrochemical gradient (A,G,+; Sahin-Toth M, Kaback HR, 1993, Protein Sci 2:1024-1033). The findings are confirmed and extended in this communication. Purified, reconstituted Val 315 + Cys permease reacts with N-ethylmaleimide or hydrophobic fluorescent maleimides but not with a membrane impermeant thiol reagent, and 0-galactosides specifically stimulate the rate of labeling. Furthermore, the reactivity of purified Val 315 + Cys permease is enhanced by imposition of a membrane potential (A*, interior negative). The results indicate that either ligand binding or A* induces a conformational change in the permease that brings the N-terminus of helix X into an environment that is more accessible from the lipid phase.
Third-Generation Biosensor for Lactose Based on Newly Discovered Cellobiose Dehydrogenase
Analytical Chemistry, 2006
The present paper describes the principle and characteristics of a biosensor for lactose based on a third-generation design involving cellobiose dehydrogenase. As resulted from a previous comparative study (submitted manuscript), the novelty of this lactose biosensor is based on highly efficient direct electron transfer between two newly discovered cellobiose dehydrogenases (CDH), from the white rot fungi Trametes villosa and Phanerochaete sordida, and a solid spectrographic graphite electrode. CDH was immobilized on the electrode surface (0.073 cm 2 ) by simple physical adsorption, and the CDH-modified electrode was next inserted into a wall-jet amperometric cell connected on-line to a flow injection setup (0.5 mL‚min -1 ). The P. sordida CDH-based lactose biosensor, proved to be the better one, has a detection limit for lactose of 1 µM, a sensitivity of 1100 µA‚mM -1 ‚cm -2 , a response time of 4 s (the time required to obtain the maximum peak current), and a linear range from 1 to 100 µM lactose (correlation coefficient 0.998). The simplicity of construction and analytical characteristics make this CDH-based lactose biosensor an excellent alternative to previous lactose biosensors reported in the literature or commercially available. The CDH-lactose sensor was used to quantify the content of lactose in pasteurized milk, buttermilk, and low-lactose milk, using the standard addition method. No effects of the samples matrixes were observed. The operational stability of the sensor was tested for 11 h by continuous injection of 100 µM lactose (290 injections). The final signal of the sensor was maintained at 98% of its initial signal, with a low standard deviation of 1.72 (RSD 2.41%).
European Food Research and Technology, 1999
In home-made sensors coimmobilizing enzymes in thin-layer plexi-cells on natural protein membranes, three enzyme cells: b-galactosidase and galactose oxidase (A), b-galactosidase and glucose oxidase (B) and b-galactosidase, galactose oxidase and glucose oxidase (C) were built into a flow-injection-analyzer system. The lactose was decomposed and oxidized by the immobilized enzymes and the hydrogen peroxide generated during the enzymatic reactions was determined by amperometric detection. The parameters for biochemical and electrochemical reactions (concentration of buffer, temperature, flow rate) were optimized in each enzyme cell. The pH optima of the lactose measurement was determined in the three enzyme cells mentioned above. The pH optimum of the cells A, B and C were 6.4, 4.5 and 4.8, respectively. The measuring ranges were 1-5 mM, 2-10 mM and 1-5 mM, while the detection limits were 0.5, 1.0 and 0.5 mM, respectively. More than 600, 1000 and 800 samples could be measured with these cells, respectively. Commercial milk and instant dessert powder products were analysed also. Our results showed that the cells B and C were more suitable for the determination of the lactose content of milk. For samples of dairy products containing added glucose, starch and other carbohydrates, enzyme cell A could be used for the efficient determination of lactose in one step.
Sensors and Actuators B: Chemical, 1992
We present a biosensor based on the transport protein lactose permease (LP) which is imbedded in a supported planar lipid bilayer (SPB) membrane, and which allows the potentiometric detection of lactose via pH measurement. Different experimental setups were used to study systematically the electric properties of the different biosensor components. In a first step the preparation of the SPB is investigated by means of high-frequency capacitancevoltage measurements. The sensor consists of an ion-sensitive field-effect transistor (ISFET) coated with the SPB membrane, the latter containing the lactose/H+ cotransporter LP from Escherichiu coli (E. coli). The sensor effect consists of a rise in the proton concentration in the small volume between the lipid membrane and the gate area upon addition of lactose to the external medium. This effect is monitored directly by the pH-sensitive ISFET.
A Lactulose Bienzyme Biosensor Based on Self-Assembled Monolayer Modified Electrodes
Electroanalysis, 2004
A bienzyme biosensor in which the enzymes b-galactosidase (b-Gal), fructose dehydrogenase (FDH), and the mediator tetrathiafulvalene (TTF) were coimmobilized by cross-linking with glutaraldehyde atop a 3-mercaptopropionic acid (MPA) self-assembled monolayer on a gold disk electrode, is reported. The working conditions selected were E app ¼ þ 0.10 V and (25 AE 1) 8C. The useful lifetime of one single TTF-b-Gal-FDH-MPA-AuE was surprisingly long, 81 days. A linear calibration plot was obtained for lactulose over the 3.0 Â 10 À5 ± 1.0 Â 10 À3 mol L À1 concentration range, with a limit of detection of 9.6 Â 10 À6 mol L À1 . The effect of potential interferents (lactose, glucose, galactose, sucrose, and ascorbic acid) on the biosensor response was evaluated. The behavior of the SAM-based biosensor in flow-injection systems in connection with amperometric detection was tested. The analytical usefulness of the biosensor was evaluated by determining lactulose in a pharmaceutical preparation containing a high lactulose concentration, and in different types of milk. Finally, the analytical characteristics of the TTF-b-Gal-FDH-MPA-AuE are critically compared with those reported for other recent enzymatic determinations of lactulose.
Time-Resolved Study of the Inner Space of Lactose Permease
Biophysical Journal, 2001
Pyranine (8-hydroxy pyrene-1,3,6-trisulfonate) is a commonly used photoacid that discharges a proton when excited to its first electronic singlet state. Follow-up of its dissociation kinetics reveals the physicochemical properties of its most immediate environment. At vanishing ionic strength the dye adsorbs to the Escherichia coli lactose permease with stoichiometry of 1:1 and an association constant of 2.5 ϫ 10 5 M Ϫ1 . The reversal of the binding at high ionic strength and the lower pK value of the bound dye imply that positive charge(s) stabilize the dye in its site. The fluorescence decay curve of the bound dye was measured by time-correlated single photon counting and the measured transient was subjected to kinetic analysis based on the geminate recombination model. The analysis indicated that the binding domain is a cleft (between 9 and 17 Å deep) characterized by low activity of water (a (water) ϭ 0.71), reduced diffusivity of protons, and enhanced electrostatic potential. The binding of pyranine and a substrate are not mutually exclusive; however, when the substrate is added, the dye-binding environment is better solvated. These properties, if attributed to the substrate-conducting pathway, may explain some of the forces operating on the substrate in the cavity. The reduced activities of the water strips the substrate from some of its solvation water molecules and replace them by direct interaction with the protein. In parallel, the lower dielectric constant enhances the binding of the proton to the protein, thus keeping a tight seal that prevents protons from diffusing.
Zeitschrift f�r Lebensmitteluntersuchung und -Forschung A, 1998
Amperometric biosensors were developed for the determination of lactose using b-galactosidase and glucose oxidase. The enzymes were co-immobilized under mild conditions in a poly(carbamoyl)sulphonatehydrogel matrix onto the surface of low-cost-screenprinted platinum working electrodes for the amperometric detection of the enzymatically generated hydrogen peroxide which was monitored at c600 mV versus Ag/ AgCl/3 M KCl. The basic sensors showed linearity over a concentration range of 0.0035-2 mM (correlation coefficient, rp0.99992). They were used in a batch system to determine the lactose content in milk. As sample pre-treatment, only dilution was necessary. Data for the determination of lactose with the enzyme electrodes were compared to those obtained using a soluble enzyme test kit (the Boehringer Mannheim UV method). The linear range of the sensors could be increased by applying additional membranes to the top of the planar sensor surface. Commercially available polycarbonate membranes with reduced pore densities and pore sizes supplied best results when fixed using doublesided tape. As an alternative approach, an extended linear range could also be accomplished by spray-coating the sensor surface using a water-based polymer dispersion.
A three-cascaded-enzymes biosensor to determine lactose concentration in raw milk
Journal of dairy science, 2000
The increasing demand for on-line measurement of milk composition directs science and industry to search for practical solutions, and biosensors may be a possibility. The specific objective of this work was to develop an electrochemical biosensor to determine lactose concentration in fresh raw milk. The sensor is based on serial reactions of three enzymes--beta-galactosidase, glucose oxidase, and horseradish peroxidase--immobilized on a glassy carbon electrode. The sequential enzymatic reactions increase the selectivity and sensitivity of the sensor. The sensor requires dilution of the raw milk and the addition of 5-aminosalicylic acid. Lactose concentrations in raw milk measured by the sensor were in good agreement with those measured by a reference laboratory using infrared technology. The results were obtained in milk samples that varied in fat and protein composition. From the results, we conclude that an electrochemical biosensor for determination of lactose concentration in fr...
Lactose Biosensor Based on Lactase and Galactose Oxidase Immobilized in Polyvinyl Formal
Artificial Cells, …, 2007
A lactose biosensor was developed by immobilizing lactase and galactose oxidase in a polyvinyl formal membrane and was attached to the oxygen electrode of a dissolved oxygen analyzer for estimation of lactose in milk and food products. The enzyme immobilized polyvinyl formal membrane was characterized by atomic force microscopy. The biosensor showed the linearity for 1-7 g dl À1 of lactose and can be reused for up to 20 measurements. The effects of pH, temperature and the stability of the immobilized lactase and galactose oxidase in PVF membrane were also studied. The enzyme membrane was found stable up to 35 C and had a shelf-life of more than three months at 4 C.