Slawomir Kalinowski

A four-electrode potentiostat-galvanostat for studies of bilayer lipid membranes

The use of four-electrode systems in the studies of electric phenomena occurring in membranes makes it possible to reduce errors caused by electrode and electrolyte resistance. A four-electrode potentiostat-galvanostat is described in this paper. It allows for electric measurements of membranes in controlled potential and current conditions or measurements of transmembrane potential. The method of selection of the operation mode of the system is described.

Programmable chronopotentiometry as a tool for the study of electroporation and resealing of pores in bilayer lipid membranes.

This paper presents the application of chronopotentiometry in the study of membrane electroporation. Chronopotentiometry with a programmable current intensity was used. The experiments were performed on planar bilayer phosphatidylcholine and cholesterol membranes formed by the Mueller-Rudin method. It was demonstrated that a constant-intensity current flow through the bilayer membranes generated voltage fluctuations during electroporation. These fluctuations (following an increase and decrease in membrane conductance) were interpreted as a result of the opening and closing of pores in membrane structures. The decrease in membrane potential to zero did not cause the pore to close immediately. The pore was maintained for about 200 s. The closing of the pore and recovery of the continuous structure of the membrane proceeded not only when the membrane potential equalled zero, but also at membrane potentials up to several tens of millivolts. The fluctuations of the pore were possible at values of membrane potential in the order of at least 100 mV. The size of the pore changed slightly and it closed after some time below this potential value.

Chronopotentiometric studies of electroporation of bilayer lipid membranes.

The constant-intensity current chronopotentiometric measurements of egg yolk phosphatidylcholine bilayer membranes (BLM) are presented. It is demonstrated that a constant-intensity current flowing through the bilayer membranes generates the pores in their structures. For the current intensity from 0.1 to 2.0 nA, the generated pores open and close cyclically. The frequency of oscillations depends on the current intensity: the higher current intensity, the higher frequency of pore oscillations. It is suggested that the presented method may allow to create one pore in BLM and to observe its dynamical behaviour. Based on chronopotentiometric curves, a method of pore conductance calculations is presented. It is demonstrated that the value of obtained conductance can be applied for pore diameter estimation. The hypothetical application of constant-current method as a biotechnological tool for selective and controlled incorporation of molecules into microorganisms is discussed.

A four-electrode system for measurement of bilayer lipid membrane capacitance

A four-electrode capacitance-to-period converter designed for capacitance measurements of bilayer lipid membranes is described. The capacitance measurement consists of cyclically charging and discharging the measured capacitance with a constant current, regarding its absolute value. The voltage of the studied capacitor is triangularly shaped. The cycle duration is proportional to the input capacitance. Capacitance measurement with a four-electrode system makes it possible to reduce considerably the errors caused by electrode and electrolyte impedance. It is possible to use high-resistance microelectrodes. The system makes it possible to measure the capacitance at an imposed polarization potential; the voltage oscillates about that value during the measurement. This makes it possible to measure the membrane capacitance as a function of polarization potential. An example is cited of using the capacitance-to-period converter in a computer-controlled measuring system.

A new system for bilayer lipid membrane capacitance measurements : method, apparatus and applications

A new method and a new apparatus for capacitance measurements on bilayer lipid membranes are described. The membrane is charged and discharged with a constant current during the measurement. The charge-discharge cycle duration, which is proportional to the membrane capacitance, is measured. The measured time period is converted into a binary number by digital systems and then this number is either further converted into a constant capacity-proportional voltage or read out by the computer. The apparatus makes it possible to measure the capacitances of voltage-polarized membranes. Application of the apparatus to capacitance measurements of bilayer lipid membranes during their potential on the capacitance is presented. The capacitances of membranes stimulated by rectangular voltage pulses and of those stimulated by a linearly varying potential were reported.

SYNTHESIS OF DIMERIC STEROIDS AS COMPONENTS OF LIPID MEMBRANES

Abstract The synthesis of three dimeric steroids 1,2, and 3, as components of artificial lipid bilayer membranes, is described. Di(3β-hydroxyfurost-5-en-26-yl) (1) was obtained from diosgenin by reductive fission of a ring F, substitution of -OH by -l, and the Wurtz reaction. Two other dimers 2 and 3 were synthesised from the pregnanoic ester 10 by an “alkylation-reduction” procedure.

A novel direct-injection photometric detector integrated with solenoid pulse-pump flow system.

A novel flow photometric detector based on paired emitter-detector diodes (PEDD), coupled with solenoid pulse micro-pumps is presented. The photometric detection chamber also plays a role of the reaction chamber. Both solutions, a sample and a reagent, are injected by solenoid micro-pumps directly and simultaneously into the chamber in countercurrent. This method ensures fast and effective mixing of the injected solutions. A good accuracy and precision of the injected volumes assure the pulse micro-pumps. This method, in comparison with other flow methods, does not require the application of the reaction coil. Thanks to this, the dispersion of the sample is minimized. The presented flow network is easy to control, miniaturize and exhibits a very low consumption of reagents and the sample. Two chemical systems were chosen and presented as models of photometric reactions: the first - Fe(III) with thiocyanate, and the second - Cr(VI) with 1,5-diphenylcarbazide. To obtain the highest repeatability, the total volume of the solutions should be smaller than the volume of the reaction-detection chamber. In this case, the whole coloured product remains in the chamber. The use of the proposed direct-injection PEDD detector considerably simplifies analytical procedures. The shape of analytical signals and their potential applications have been discussed.

Changes of structural and dynamic properties of model lipid membranes induced by α-tocopherol: implication to the membrane stabilization under external electric field

The effects of alpha-tocopherol on electric properties of bilayer lipid membranes were investigated. Planar bilayer membranes formed by the Mueller-Rudin method were used. Voltammetric and chronopotentiometric measurements were performed using a four-electrode potentiostat-galvanostat. It was demonstrated that registration of membrane capacitance, resistance, and voltammetric characteristics provided information about the change in the structure and permeability of bilayer lipid membranes. The results suggested that incorporation of alpha-tocopherol into lipid membrane destabilized its structure and facilitated the electrogeneration of pores. The possible role of observed changes in physiological functions of alpha-tocopherol was discussed.

Application of direct-injection detector integrated with the multi-pumping flow system to photometric stop-flow determination of total iron

A novel direct-injection detector (DID) integrated with multi-pumping flow system (MPFS) for the photometric determination of iron is proposed. Paired emitter-detector diodes have been used as a photometric detection system. The sample and reagent were injected using appropriate solenoid pulse micro-pumps directly into the detection chamber where effective mixing occured. The use of proposed stop-flow detector considerably simplified the analytical procedure. The potassium thiocyanate has been chosen as a chromogenic reagent for photometric Fe(III) detection. The total volume of reagent and sample/standard solutions involved in the detection process was adjusted to the volume of the reaction-detection chamber. Calibration graph was found to be linear in the range up to 10mgL(-1). The detection limit (3s(b)/S) was 0.15mgL(-1). The repeatability (R.S.D.), calculated from 10 analyses of sample containing 5mgL(-1) Fe(III), was 1.5% and the sample throughput 180 determinations per hour. The consumption of sample and reagent was 20μL each with the waste generation at the level of 0.24mL. The applicability of the proposed method to the determination of total iron in groundwater samples has been proved. The analytical parameters are compared to those obtained exploiting the MPFS system with typical configuration containing a confluence point and reaction coil.

A study of the deposition of iron on mercury and glassy carbon electrodes

Abstract The processes of reduction and reoxidation of iron(II) in thiocyanate, chloride and perchlorate media have been investigated by cyclic voltammetry and chronopotentiometry on mercury (HMDE) and glassy carbon (GCE) electrodes and interpreted as follows. On mercury electrodes, iron deposited from aquo and chloride complexes of Fe(II) is wetted by mercury and penetrates into the bulk of the electrode. On the other hand, FeS formed during the electroreduction of thiocyanate complexes of Fe(II), is adsorbed on the electrode surface and makes the wetting of iron by mercury impossible. Consequently, the iron stays on the electrode surface and accelerates the process of hydrogen evolution. This, in turn, leads to alkalization of the solution and to precipitation of Fe(OH) 2 in the pre-electrode layer. Subsequently, both FeS and Fe(OH) 2 catalyze the formation of metallic iron on the mercury surface. FeS and Fe(OH) 2 are also formed in the surface layer during the reduction of thiocyanate complexes of Fe(II) on the GCE. In practice, neither substance participates in the initiation of iron settling on the glassy carbon surface, but their influence on the electrode processes of systems examined on the GCE is significant.