Patent Application: US-48135195-A

Abstract:
current fluctuations typical of those observed with biological channels which are ion selective and inhibited by divalent cations and protons have been observed in synthetic membranes , and a process for controlling permeability of a synthetic membrane to ions and uncharged molecules may be used in a switch or sensor , for example to monitor the content of a solution , to trigger macroscopic events by local microscopic changes and to alter the ionic content of a solution e . g . desalination .

Description:
the term &# 34 ; ionic solution &# 34 ; in this context refers to a solution containing ions which may be e . g . a solution of an ionic material , such as a salt , dissolved in an appropriate solvent , or may be simply a liquid , such as water , in which a proportion of the liquid exists in the form of ions . suitable ions which may be transported through the membrane pore include alkali metal ions ( e . g . li + , na + , k + , rb + ), halide ions ( e . g . f - , cl - , br - , i - ) and multivalent ions ( e . g . mg 2 + , ca 2 + , so 4 2 - , phosphate ). preferred ions will depend on the use to which the process is being applied e . g . desalination . suitable solute molecules which may be transported include water soluble molecules whose effective size is less than the effective cross - sectional area or width of the pore ( e . g . sugars such as glucose , amino acids , certain hormones , neurotransmitters , drugs , immune molecules or polymers such as polyethylene glycol ). current switching behaviour typical of that observed in an ionic channel in a naturally occurring membrane may be reproduced by a synthetic membrane containing narrow pores . the membrane may be a plastic film such as a polycarbonate or other polyester , e . g . polyethylene terephthalate ( petp ). the applicants have investigated petp films of 5 or 10 micrometers thickness and found them to display the effect of the invention , but thinner or thicker films could be used , depending on the requirements of the use to which the process is being applied . for example , the minimum thickness may depend inter alia on physical characteristics such as the ability of the film to be handled by the manufacturer or user and on the pressure the film is subject to during use . for electrical devices ( e . g . sensors ) thin films are likely to prove optimal . for flow devices ( e . g . desalination ) thick films are likely to prove optimal ; alternatively laminar flow between two apposed membranes may be used . the applicants believe that other synthetic membranes exposed to the track - etch procedure described below are also suitable for use in the present process ; these include polymers that are positively charged as well as those like petp that are negatively charged after track etching . in the case of positively charged membranes , some of the properties e . g . selectivity and inhibition by protons can be reversed . narrow pores in plastic films such as polyester , e . g . petp , may be formed by irradiating the film with heavy ions such as 132 xe , 129 xe , 84 kr or 59 co accelerated in a cyclotron , to produce tracks ; the polymer is then treated with a hot alkali 3 ! to hydrolyse parts of the polymer thereby causing local solubilization of the film material particularly in the regions affected by the heavy ion irradiation . the whole process is called track - etching . in the case of laminar flow between two apposed membranes track - etching of the membrane in order to create narrow pores is unnecessary , but chemical treatment e . g . etching alone of the surfaces may be required . in the track - etching process cylindrical or conical pores are formed which penetrate the film . the pore diameter depends on the energy and species of heavy ions used for the film irradiation , on the type of polymer used and especially on the conditions of the etching process . to achieve membranes which may display the effect of the present invention , beams of heavy ions may be used which typically have the following energies : 132 xe ( 121 mev ), 129 xe ( 124 mev ), 84 kr ( 74 mev ) and 59 co ( 68 mev ), with an ion fluence of from 6 × 10 5 to 1 × 10 11 cm - 2 . appropriate alkalis and temperatures for the use in this method would be evident to the ordinarily skilled person , and for materials such as petp include e . g . 0 . 1m naoh , 1 . 0m k 2 co 3 and 2 . 5m k 2 co 3 used at a temperature of about 80 ° c . for the purposes of the present invention the pore diameter or width should be less than 20 nanometers . the chemical etching of pores into a material such as polyethylene terephthalate results in hydrolysis of the petp generating free carboxyl groups making the pores in the membrane more hydrophilic , but other possible membrane materials could be used . the density of pores in the membranes that the applicants have studied so far range from 1 to 5 × 10 9 pores per cm 2 but there is no reason to suppose that higher pore densities could not also be used . the properties of individual pores are independent of pore density . for electrical devices ( e . g . sensors ) the pore density should be low . for flow devices ( e . g . desalination ) the pore density should be high ; an alternative mode in the latter case is for flow to be between two closely apposed membranes such that the space between them is less than 20 nm . transfer of ions , i . e . ion current across such membranes may exhibit rapid switching from a high conductivity to a low conductivity state , even when the voltage across the membrane is held fixed . the rapid switching shown by such a narrow pore membrane resembles the rapid switching shown by biological ion channels . this switching may be observed whenever the ratio of surface area to volume of the conducting phase is high , such as in narrow pores or in the space between two closely apposed solid surfaces or lipid monolayers . switching can be modified and controlled by controlling the amount of particular ions such as protons and multivalent ions in solution . this control may be achieved by adding protons or a source of multivalent ions , and may be reversed by adding alkali to remove such protons or a sequestrant such as ethylene diamine tetraacetic acid ( edta ) to remove multivalent ions . the control of conduction through the narrow pore includes control of the transfer of water and uncharged molecules as well as ions . fig1 a : shows how current varies as a function of applied potential for two filters . fig1 b : shows how conductivity varies in the presence of a solution of polyethylene glycol of varying molecular weight . fig2 : shows the dependence of pore conductance on ionic strength as a double logarithmic plot . fig3 a : shows representative current fluctuations across filters as a function of time . fig3 b : shows the amplitude distribution histograms of the current in the predominant conducting states . fig3 c : shows time distribution histograms for the high and low conducting states . fig4 a and 4b : shows how the conductivity varies with the concentration of dilvent cation or proton . fig4 c : shows how the logarithm of the ratio of the time constants for the high and low conducting states varies with the concentration of divalent cation or proton . fig5 : shows the apparatus used to monitor ion currents through one or a few pores in a track - etched polyethylene terephthalate ( petp ) filter containing many pores . fig6 a and b : shows the frequency distribution of ionic conductivity ( a ) and calculated diameter ( b ) of pores in a track - etched petp filter . fig8 : shows ionic selectivity of pores in track - etched , petp filters and in such filters coated with pmvp . the filters used were approximately 5 μm thick and made of polyethylene terephthalate ( petp ; &# 34 ; lavsan &# 34 ;). after heavy ion bombardment to induce nuclear tracks , the filters were &# 34 ; etched &# 34 ; with hot alkali ; this treatment breaks ( hydrolyses ) some of the ester bonds generating free carboxyl groups . these may be negatively charged and make the filters more hydrophilic 3 !. two separate filters , having slightly different apparent pore sizes were used . they are referred to throughout as filter 1 and filter 2 . the pore density for each filter was approximately 1 pore / cm 2 . filters were clamped between two small chambers ( volume 0 . 3 ml each ) which contained kcl ( 0 . 1 - 3m ), buffered with 0 . 005m hepes ph 7 . 4 , respectively . current was measured at room temperature with ag / agcl electrodes monitored with a virtual grounded operational amplifier with a feed - back resistor of 10 9 ohms . the membrane switches between a high conducting or &# 34 ; on &# 34 ; state and a low conducting or &# 34 ; off &# 34 ; state . the reversal potential , i . e . the sign and magnitude of the voltage ( ψ ) at zero current was used to deduce the selectivity of the filters for cations over anions , in the presence of a five - fold gradient of kcl . the modal current in the high conducting state is obtained from amplitude histograms as described hereafter . in fig1 a this current is plotted as a function of applied potential for filter 1 (□) and filter 2 (). the selectivity ( t + ) was calculated from the reversal potential ( ψ ) using the equation ( i ): ## equ1 ## the terms &# 34 ; trans &# 34 ; and &# 34 ; cis &# 34 ; are used to identify the compartments on either side of the membrane . the experiments illustrated in fig1 a show the reversal potential to be 35 mv for each filter , indicating a selectivity value for cations over anions ( t + ) of 0 . 93 . the relative pore sizes of the two filters are estimated by assessing the effect on conductivity of solutions of a non - electrolyte , polyethylene glycol ( peg ), of varying molecular weight . if the non - electrolyte is able to pass through the pore , conductivity is decreased ; if not , it is unaffected . by determining the size of the non - electrolyte that is unable to pass through the filter , an estimate of its average pore size can be obtained . the conductivity of the pore in the presence or absence of 20 % ( v / v ) polyethylene glycol ( peg ) of differing molecular weights is compared . the conductances ( g ) are calculated from the modal current of the highest conducting state at a potential of 0 . 2v for each filter in the presence of 20 % peg relative to that in the absence of peg . the conductances for filter 1 (□) and filter 2 () are shown in fig1 b and expressed as set out in equation ii as the ratio ( a ) of that parameter with the same parameter but measured in the absence of the filter : ## equ2 ## the approximate size of peg which results in &# 34 ; cut off &# 34 ; is considered to be the size which yields a value of a of 0 . 5 . this size was found at molecular weights of ˜ 3000 for filter 1 and ˜ 1000 for filter 2 . based on the &# 34 ; exclusion &# 34 ; volume of pegs in water , these correspond to pore radii of about 1 . 4 nm for filter 1 and 1 nm for filter 2 . it is seen that the pore in filter 2 appears to be somewhat narrower than that in filter 1 , compatible with a lower conductance ( fig1 a ). the dependence of pore conductance on ionic strength is shown in fig2 as a double logarithmic plot . identical solutions containing kcl at the concentration indicated in fig2 with 10 - 4 - 3 × 10 - 3 m edta and 0 . 005m hepes at ph 11 (♦), 8 . 3 (), 7 . 5 (▴) or 6 . 5 (▪) bathed each face of filter 2 . conductance ( g ) of the highest conducting state at an applied potential of 0 . 8v is shown . similar data were obtained at an applied potential of 0 . 2v , except that at ph 6 . 5 the high conducting state was not observed . at ph 11 conductance is almost independent of ionic strength for current ( i )≦ 0 . 1 . at lower ph values the conductance has a linear dependence on i but with a slope much less than unity . these results suggest that negative charges either in the pore or close to its mouth exert considerable influence on the conductance by causing the local cation concentration greatly to exceed that in the bulk solution . the change of slope over the range of ph values tested suggests that the negative charges titrate with an effective pk around neutrality . current fluctuations are maximal around this ph , with smaller , fewer fluctuations above or below it 4 !. such an effect has recently been observed with s . aureus α - toxin induced pores across lipid bilayers also 5 !. current fluctuations across the filters were measured and typical records of current are shown in fig3 a . filter 1 was bathed in 0 . 1m kcl , 0 . 005m hepes ph 7 . 4 without ( i ) or with 3 . 10 - 4 m ( ii ) or 3 . 10 - 3 m cacl 2 ( iii ); filter 2 ( iv ) was bathed in a similar medium without cacl 2 at ph 8 . 0 ; currents at 0 . 2v were recorded on videotape using a biologic pcm instrumentation recorder and subsequently analysed using cambridge electronic design patch and voltage - clamp software . fig3 a shows representative traces . discrete changes between conducting and non - conducting states are seen . analysis of the two predominant states ( fig3 b ) shows the higher one to be 11 pa and the lower one 2 . 5 pa for filter 1 . for filter 2 the values are 3 . 3 pa and 1 . 2 pa . the lower state is unlikely to represent a &# 34 ; leak &# 34 ; current , as ( a ) it changes on the addition of ca 2 + ( see below ), ( b ) it disappears altogether at low ph and ( c ) it is not seen if the filter is moved so that there is no pore at all between the two chambers . fig3 c presents time distribution histograms for the high and low conducting states respectively together with the time constants for each state obtained by fitting a single exponential to the distribution . for filter 1 mean values (± s . d .) of the time constant for the high conducting state ( τ h ) of 210 ± 59 ms and of the low conducting state ( τ l ) of 23 ± 8 . 7 ms ( n = 8 ) were found . the difference between the high and low conductance states varies with voltage and ionic strength . the amplitude of the difference between the current of the high and low conducting states as a function of applied voltage or of the ionic concentration of the bathing medium are shown in table 1 below ; n . d . indicates a value was not determined . table 1______________________________________difference in current between the high and low conductingstates of filter 2 at different potentials and ionic strengths . current / paapplied potential / v 0 . 1m kcl 0 . 2m kcl______________________________________0 . 2 2 . 7 4 . 00 . 4 5 . 5 7 . 00 . 6 8 . 8 n . d . ______________________________________ although the absolute value of the higher conductance state depends somewhat on the pre - treatment of the filter prior to use ( e . g . exposure to ethanol , lipids , other chemicals , etc ) the difference in amplitude between the higher conductance state and the lower one is remarkably constant . other factors , such as ph ( fig2 ) or the presence of divalent cations ( see below ) do affect both conductance states . on the addition of 0 . 3 mm ca 2 + , for example , the difference between the higher and lower conducting state is decreased to 9 . 5 and 3 . 5 pa respectively for filter 1 ( fig3 b ). such intermediate amplitudes also become detectable in the absence of ca 2 + if a long enough recording is analysed . addition of 3 mm ca 2 + abolishes the high conducting state completely ; high conductance is restored by the addition of edta ( not shown ). similar effects are observed by addition of other divalent cations such as zn 2 + or mg 2 + , or by reduction of ph . the relative efficacy of divalent cations and protons at reducing current is shown in fig4 . filter 1 was bathed in 0 . 1m kcl ( fig4 a ) or 3m kcl ( fig4 b ) with 0 . 005m hepes , initial ph 7 . 4 , h + / oh - ( o ) or divalent cations ( at ph 7 . 4 : zn 2 + (▴), ca 2 + (▪), mg 2 + (♦)) were added to give the final concentrations indicated . the time - averaged conductance ( g ) at 0 . 2v is expressed relative to the maximum observed conductance ( g o ) at the start of each titration . fig4 c presents the logarithm of the ratio of the time constants for the high ( τ h ) and low ( τ l ) conducting states obtained at 0 . 2v in 0 . 1m kcl , 0 . 005m hepes at the ph specified (□-- filter 1 ; ◯-- filter 2 ) or in a similar medium at ph 7 . 4 ( filter 1 ) containing the concentration of cacl . sub . (▪) or znso 4 (▴) indicated ; error bars represent the standard deviation found for filter 1 ( n = 8 ) at ph 7 . 4 and for filter 2 ( n = 4 ) at ph 8 . 0 . it is seen from fig4 a and 4b that in both cases h + & gt ; zn 2 + & gt ; ca 2 + ≧ mg 2 + with 50 % inhibition at molar concentrations of approximately 10 - 7 . 7 , 10 - 4 . 5 , 10 - 3 . 4 , and 10 - 3 . 0 ( 0 . 1m kcl ) and 10 - 7 . 5 , 10 - 2 . 8 , 10 - 1 . 7 and 10 - 1 . 7 ( 3m kcl ) respectively for filter 1 , and 10 - 8 . 4 , 10 - 6 . 0 , 10 - 4 . 2 , and 10 - 4 . 2 ( 0 . 1m kcl ) for filter 2 ( not shown ). the fact that inhibition is little different in 0 . 1m and 3m kcl suggests that simple screening of surface charge plays at best a minor role . the data points in fig4 a and 4b are derived from time - averaged conductances . kinetic analysis ( as in fig3 c ) of fluctuations observed in the presence of divalent cations and protons is shown in fig4 c . both sets of data show that filter 2 is more sensitive to inhibition by divalent cations and protons , again compatible with a smaller apparent pore size ( see fig1 b ). ionic conduction in narrow pores may well be similar to that which occurs in some hydrated zeolites where it can be shown 6 ! that only the bare ion moves , although it is at all times hydrated by an essentially static array of water molecules . calculation 7 ! shows that the centres of the planar pentagon and puckered hexagon water rings that preserve tetrahedral bonding can provide low energy ion binding sites . the origin of the rapid switching maybe connected with the fact that lattice sums 8 ! and molecular dynamic simulations 9 ! show that water in narrow pores will be electrically ordered . changes in order could lead to switching 8 ! due to the powerful electric fields generated . an alternative explanation of the switching is that changes in the nature of the ions absorbed on the pore walls alter the water structure near the pore walls and hence the pore conductance . in zeolites it is known 10 ! that exchange between cations in the bathing solution and the alumina - silicate cage can lead to phase changes in the cages and hence of the water contained within them . such metastability may account for rapid transitions in the conductance through narrow pores . in a biological system the channel protein must also supply a gating mechanism sensitive to the membrane voltage 11 ! and to the presence of particular ligands 2 !, which may be in series with the water - pore gating described here . in addition the protein must distinguish between different cations 12 ! and carry out other activities such as inactivation 13 !. individual pores in track - etched petp filters containing up to 5 × 10 9 pores cm - 2 also exhibit selective ion flow and rapid switching between discrete conductance states provided that their overall ionic conductivity is sufficiently low ( less than 100 ps in 0 . 1m kcl ). such pores can be studied with glass micropipettes ( 1 ) having a tip diameter of about 1 μm using the apparatus illustrated in fig5 ; ion currents through the pores in the filter ( between bath and pipette ) are monitored with ag / agcl electrodes in the electronic circuit shown . when bath and pipette each contain 1m kcl with 0 . 005m hepes , ph adjusted to 7 . 4 with koh the conductance recorded each time the pipette contacts the filter falls in the range 5 to 2000 ps as shown in fig6 ( left hand panel ). assuming that each conductance represents that through a cylindrical pore in a 10 μm thick filter , the diameter of that pore can be calculated and the results of such calculations are shown in fig6 right hand panel . pore diameters exhibit an approximately normal distribution with a mean close to 200 å . rapid switching between high and low conducting states is observed when the bath and pipette contain 0 . 1m kcl with 0 . 005m hepes , ph adjusted to 7 . 4 with koh and the overall pore conductance does not exceed 100 ps . typical results at positive or negative applied potentials are shown for four different pores in fig7 . selectivity of ion currents through individual pores using the apparatus shown in fig5 was assessed from the reversal potential ( the applied potential at which no ion current flows ) observed when the pipette contained 1m and the bath 0 . 2m kcl each with 0 . 005m hepes , ph adjusted to 7 . 4 with koh . cation selectivity , indicated by transference numbers ( t + ) in excess of 0 . 5 , is observed for pores in track - etched petp filters whereas anion selectivity ( t + less than 0 . 5 ) is observed in similar filters coated with polyvinylmethylpyridine ( pvmp ) to confer positive surface charge on the petp . in either case pores with conductances greater than 1000 ps show little selectivity ( t + ≈ 0 . 5 ); selectivity increases , for cations with petp filters and for anions with pvmp coated filters , progressively as pore conductance diminishes ( fig8 ). 2 . hille b . ionic channels of excitable membranes . sinauer associates inc ., sunderland , mass . ( 1984 ) pp 277 , 303 - 353 . 3 . apel p y , didyk a , kravels l i and kuznetsov v i nucl . tracks radiat . meas . 17 191 - 193 ( 1990 ). 4 . pasternak c a , bashford c l , korchev y e , rostovtseva t k and lev a a . 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