Patent Application: US-201615000155-A

Abstract:
an electrode assembly that may be used , for example , for electrochemically analysing a sample to determine the presence of a species having biomembrane activity comprises at least one working electrode comprised of a conductive carrier substrate having a surface coated with mercury immobilised on the surface of the substrate . the surface of the mercury remote from said substrate is coated with a phospholipid layer . the preferred carrier substrate is platinum . the electrode assembly may be incorporated in a flow cell .

Description:
referring firstly to fig1 , an electrode assembly 1 in accordance with the invention comprises a working electrode 2 which is comprised of the combination of an iridium layer 3 having a surface coating of mercury 4 on which is deposited a phospolipid monolayer 5 . as illustrated in fig1 , the mercury layer 4 is located within a well structure ( for which iridium layer 3 provides a basal surface ) formed in an upper silica layer 6 , the well being circular with a selected diameter in the range 2 μm to 10 μm and having a depth of 0 . 5 μm . the mercury occupies the full cross - section of the well structure whereby there is no exposed free iridium . additional principal features of the illustrated electrode assembly are a lower silicon substrate 7 with a silica surface and a gold conducting layer 8 which is a electrically conducting relationship with iridium layer 3 . provided between the silicon / silica layer 7 and gold layer 8 is a titanium adhesion layer 9 and similar adhesion layers 10 and 11 are provided respectively ( i ) between the iridium layer 3 and gold layer 8 , and ( ii ) between iridium layer 3 and upper insulating layer 6 . reference is now made to fig2 which is a perspective view of a microelectrode array based on the structure illustrated in fig1 but with the titanium adhesion layers 9 - 11 omitted for the purposes of clarity and the well structures ( referenced in fig2 as 12 ) being shown as “ empty ” ( i . e ., without the mercury layer 4 and its associated phospholipid monolayer 5 ). fig2 does however also illustrate circular and part - circular reference electrodes 13 provided on the upper insulating layer 6 , each electrode 13 having a radius of 50 μm and each being centred at a respective well 12 . fig3 illustrates a step wise procedure for producing an electrode assembly of the type illustrated in fig2 . the illustrated procedure starts with a 10 cm × 10 cm silicon wafer with a 90 nm sio 2 surface layer ( e . g ., as available from idb technologies ). this provides layer 7 for the structure illustrated in fig1 and 2 . there is then deposited in succession on to the sio 2 surface layer ( a ) the titanium adhesion layer 9 ( 30 nm ), ( b ) the gold conduction layer 8 ( 100 nm ), ( c ) the titanium adhesion layer 10 ( 30 nm ), ( d ) the iridium layer 3 ( 30 nm ) and ( e ) the titanium adhesion layer 11 ( 30 nm ). all of these layers may be deposited by e - beam evaporation . it should be noted that , at this stage , the titanium adhesion layer 11 is deposited as a continuous layer . the resulting structure is depicted in fig3 a . in the next step of the process , a 500 nm sio 2 layer is deposited on titanium adhesion layer 11 by means of low - temperature plasma enhanced chemical vapour deposition ( pecvd ) to provide layer 6 ( but not having , at this stage , the wells 12 formed therein ). the product at this stage is shown in fig3 b . for the next step of the process , a positive resist material is spun on to the sio 2 layer 6 then baked at 150 ° c . and washed in chlorobenzene to provide a “ hardened ” surface resist layer depicted by reference numeral 14 ( see fig3 c ). a mask 15 incorporating the pattern for the silver rings 13 is positioned over resist layer 14 which is then patterned using photo / e - beam lithography ( fig3 d ). mask 15 is now removed and next resist layer 14 is chemically etched down to the sio 2 layer 6 ( fig3 e ). in the step illustrated in fig3 f a 100 nm silver layer 16 is evaporated on to the surface of the resist layer 14 but , more importantly , also within the channels of the pattern of circles and arcs defined therein . subsequently resist layer 14 is removed to leave a structure as illustrated in fig3 g in which the upper surface has circular and part circular traces 17 . although not illustrated in the drawings , procedures generally along the lines described for fig3 c - f may be repeated to provide a platinum counter electrode on the silica layer 6 . a further resist layer 18 capable of withstanding plasma etching is now applied to silica layer 6 ( and overlays the silver traces 17 ) and plasma etched to form a pattern of circular apertures 19 , that expose the iridium layer ( fig3 h ). in other words , the etching is through the sio 2 layer 6 and also through the titanium adhesion layer 11 . if desired , a 10 nm gold wetting layer may be deposited at this stage to the exposed iridium surfaces at the bases of the apertures 19 . removal of the resist 18 produces the structure illustrated in fig3 i in which wells 12 ( corresponding to those in fig2 ) have been formed . the silver trace 12 may be anodised in a chloride rich solution to produce a stable ag / ag / cl reference electrode with fast kinetics and a stable reference potential . the layer of mercury 4 ( described with reference to fig1 ) may be electrodeposited ( as described above ) on to those portions of the iridium layer 3 exposed at the base of the wells . subsequently the phospholipid layer may be deposited , again using techniques as described above . fig4 illustrates a particular embodiment of microelectrode array in accordance with the invention . the illustrated electrode assembly 100 comprises a 10 × 10 array of working electrodes 101 produced in the manner described more fully above with reference to fig3 and thus comprising mercury coated iridium provided with a phospholipid layer on the mercury surface , the electrodes 101 being associated with a conducting trace 102 . the assembly further comprises a ag / ag / cl reference electrode ( not individually referenced but similar to that shown in fig2 ) associated with a conducting trace 103 . a platinum electrode 104 associated with a conducting trace 105 is also provided . conducting traces 102 , 103 and 105 are associated with respective gold contact pads 106 , 107 and 108 respectively for connection to electronic control / measurement systems , e . g . as shown schematically in fig5 . although the construction of the sensor has been described with specific reference to iridium as the carrier metal , it will be appreciated that the same principles of construction may be employed for the other carrier metals that may be used in accordance with the invention ( i . e ., palladium , platinum and tantalum ). reference is made to fig8 which is a schematic cross - sectional view to a much enlarged scale of a composite pt / hg electrode assembly 200 on which a phospholipid layer may be deposited . electrode assembly 200 comprises a silicon wafer 201 on which is deposited a layer 202 of silicon nitride ( e . g ., having a thickness of 500 nm ). formed in the silicon nitride is at least one well on the base of which is an adhesion layer ( e . g ., 30 nm ) of titanium 203 on which is deposited a 100 nm thick layer of platinum 204 . filling the well is a layer of mercury 205 provided on its upper surface ( as viewed in fig8 ) with a monolayer 206 of a phospholipid . reference numeral 207 in fig8 represents electrolyte solution in which the electrode would , in use , be immersed . although not illustrated in fig8 , platinum layer 204 is associated with a conductive trace for connection to a potentiostat . fig9 illustrates an electrode assembly 300 constructed in accordance with the general principles shown in , and described with reference to , fig8 above . the electrode assembly 300 is generally rectangular and is formed towards one end thereof with two working electrodes 301 and 302 each within a well of a silicon nitride layer 303 . towards the opposite end of electrode assembly 301 are two contact pads 304 and 305 . electrode 301 is connected by a conductive trace 306 to contact pad 304 whereas electrode 302 is connected by conductive trace 307 to contact pad 305 . referring now to fig1 a - c there are illustrated schematic views of one embodiment of flow cell 400 constructed for the purposes of proof - of principle ” ( see example 7 below ) and incorporating an electrode assembly 300 of the type described with reference to fig9 . the schematic drawings of 10 a - c respectively illustrate side , end and plan views of the flow cell 400 which ( particularly from fig1 a and b will be seen to comprise a base portion 401 and a top portion 402 both formed in the manner described more fully below . upper surface of base portion 401 is formed with a rebate such that electrode assembly 300 may sit therein so that its end provided with the contact pads 304 and 305 projects from the flow cell ( see particularly fig1 b ). it should be understand from fig1 b that , in the illustrated position of the electrode assembly 300 , the electrodes 301 and 302 as well as the contact pads 304 and 305 are uppermost . the under surface of upper portion 402 has a central recess which ( with upper portion 401 and lower portion 402 assembled together in the manner illustrated in fig1 a ) forms a “ measurement cell ” 403 into which the electrodes 301 and 302 face . leading to the left from measurement cell 403 ( as viewed in fig1 a ) is an electrolyte entry channel 404 and leading to the right is an electrolyte outlet channel 405 . communicating with electrolyte inlet channel 404 is an injection port 406 for a sample to be analysed whereas leading from electrolyte outlet channel 404 are bores 407 and 408 , one for incorporating a ag / agcl reference electrode and the other a pt auxiliary electrode ( neither shown ) separately from the electrode assembly 401 . an annular groove is formed in the under surface of upper portion 402 of flow cell 400 and receives a sealing o - ring 409 . although not illustrated in the drawings , screw holes are provided for receiving screws to assemble upper and lower portions 41 and 42 together . each of the electrodes 301 and 302 may have a different phospholipid deposited therein . for measurement purposes the working electrodes 301 and 302 as well as the auxiliary and reference electrodes are connected to a potentiostat in the manner illustrated in fig5 . the arrangement will be such that electrodes 301 and 302 can be individually addressed . in use of the flow cell , electrolyte is passed into inlet 404 and allowed to flow through “ measurement well ” 403 and then through outlet channel 405 . sample to be analysed may be injected into part 406 . individual measurements may then be made for the effect of the sample on different phospholipids on electrodes 301 and 302 . to establish proof of principle , mercury was electrodeposited on ir circular discs surrounded by glass as an insulator . the electrodes were washed with deionised water and a phospholipid was then deposited on the mercury layer by passing the mercury coated electrode through a film of a solution of dioleoyl phosphatidycholine ( dopc ) in pentane at an electrolyte - gas interface . subsequently the pentane was evaporated to leave the dopc on the mercury surface . the phospholipid layers were monitored by rapid cyclic voltammetry ( rcv ) at 80 v s − 1 . a comparative experiment was conducted using dopc on a hanging drop mercury electrode ( prior art ). the results are shown in fig6 a for which the lighter trace shows the results for the mercury coated iridium electrode whereas the darker trace is for the hmde electrode . it will be seen from fig6 a that the two traces are virtually identical and both demonstrate the characteristic peaks ( 1 and 2 ). fig6 b shows the results for the mercury coated iridium electrode scanned at 100 v s − 1 . once again the two characteristic peaks ( 1 and 2 ) can clearly be seen . the results show that the mercury coated electrode shows sharper voltammetric peaks at a rapid scan rate . this finding indicates that the occurrence of the phase transitions is a function of electrode size . sharper voltammetric peaks are more suitable for analytical purposes and favour the microelectrode as a support for phospholipids . we have shown that removal of phospholipid from the microelectrode surface can be achieved by applying an extreme potential of − 3 . 0 v vs ag / agcl . the voltammetric peaks displayed in the rcv s in fig6 a and b can be used for the selective analytical recognition of a large number of dissolved organic species at very low ( nano to μmol dm − 3 ) level . these results show that stable ordered phospholipid layers can be deposited on to hg / ir microelectrodes . mercury was electrodeposited on a close packed hexagonal array of 1800 platinum micro electrodes ( on a base of pt of 2 mm diameter ), the microelectrodes being of dimension 10 μm diameter with a 20 μm spacing centre - to - centre . phospholipid dopc was deposited on this array of microelectrodes by evaporating a solution of phospholipid on the surface of the array . the array was introduced into an electrolyte solution where it was voltammetrically cycled between potentials of − 0 . 2 and − 2 . 0 v vs . ag / agcl . this annealed the dopc monolayer to form a stable organised layer as demonstrated by fig7 which is a cyclic voltammetry plot at 30 v s − 1 from − 0 . 2 v to − 1 . 0 v . this plot displays the characteristic phase transitions . these layers have been shown to be stable for at least one hour . electrode assemblies of the type illustrated in fig9 was prepared using the procedure set out below to produce an assembly in which the wells in the silicon nitride layer 303 at a diameter of 960 μm . a 100 mm thick silicon wafer substrate was cleaned with piranha solution ( a 2 : 1 ( v : v ) mixture of sulphuric acid and hydrogen peroxide ) and subjected to thermal oxidation to grow a layer of dense oxide on the wafer surface . standard uv photolithography techniques were used to produce a plurality of identical resist patterns on the wafer each corresponding to one electrode assembly . the individual resist patterns had developed positive resist ( absent resist ) at the regions corresponding to the working electrodes 301 and 302 , the contact pads 304 and 305 and the conductive traces 306 and 307 . using conventional thermal evaporation techniques a 30 nm thick titanium adhesion layer and then a 100 nm thick platinum layer were applied to the substrate ( see layers 203 and 204 in fig8 ). the pattern was revealed using the standard practice of “ metal lift - off ” by dissolving the photo - resist in acetone . a layer of silicon nitride approximately 500 nm thick was then deposited using plasma enhanced chemical vapour deposition ( pecvd ) ( see layer 202 in fig8 ). further uv photolithography was then used to pattern the electrode regions 301 and 302 and the contact pads 304 and 305 ( resist developed selectively in these regions ) using a second photo mask ( etch mask ). the underlying silicon nitride was then etched using a hydrofluoric acid based wet etch down to the surface of the platinum layer , so the latter was exposed at the base of wells in the silicon nitride and to provide the contact pads 304 and 305 . the remaining resist was then removed and the device cleaned with piranha solution . individual electrode assemblies were subsequently isolated from the others formed on the wafer by dicing the wafer using a wafer saw . electrodeposition of mercury onto platinum disc working electrodes was performed in a standard three electrode cell containing a double junction reference electrode ( 3 . 5 mol dm − 3 kcl , ag / agcl inner filling , 0 . 1 mol dm − 3 perchloric acid outer filling ) and a platinum bar auxillary electrode both supplied by metrohm . the working electrodes were introduced into the cell by means of a micromanipulator and connected via crocodile clips attached to platinum bond pads . the potential at the surface of the working electrodes was set using a pgstat12 ( ecochemie , utrecht , the netherlands ) potentiostat controlled by autolab software . the silicon wafer based working electrodes were cleaned prior to electrodeposition in a hot solution of sulphuric acid ( fisher scientific ) and 30 % hydrogen peroxide ( fluka ) in a ratio of approximately 3 : 1 before drying under nitrogen . electrodeposition was performed at − 0 . 4v vs . the 3 . 5 mol dm − 3 kcl ag / agcl reference and monitored by means of chronocoulometry . the deposition was terminated by opening the circuit and immediately removing of the electrode from the deposition solution once 1 coulomb of charge had passed . the liquid mercury deposited on the platinum was in the form of a “ flattened hemisphere ”. the mercury was immobilised sufficiently on the platinum to allow the electrode assembly to be turned “ upside down ” without loss of mercury . this example demonstrates the stability of an electrode assembly produced in accordance with the procedure of example 3 . the electrode assembly was tested in a three electrode cell which was temperature controlled at 25 ° c . and which contained 0 . 1 mol · dm − 3 kcl phosphate buffered at ph 7 . 4 . the solution was deaerated prior to introduction of the electrode assembly by bubbling argon gas through the stirred solution . the electrode assembly was lowered into the cell using a micro manipulator and connected to the external potentiostat by crocodile clips attached to one of the electrode contact pads . the working electrode potential was set relative to a ag / agcl 3 . 5 mol · dm − 3 kcl reference electrode separated from the cell by a porous glass frit . a platinum bar counter electrode completed the circuit . rapid cyclic voltammetry measurements were carried out using an acm research potentiostat ( acm instruments , cumbria , uk ) interfaced to a powerlab 4 / 25 signal generator and adc ( ad instruments , oxfordshire , uk ) controlled by scope ™ software . the electrode assembly was washed with a jet of mili q water before introduction into the cell . it was then electrochemically cleaned for 30 seconds using the procedure described in the following paragraph . for the cleaning procedure , the potential of the working electrode was scanned in a cathodic direction rapidly desorbing any contaminating organic material into the bulk solution using the following conditions : ( 5 single consecutive sweeps recorded + a sweep recorded after repetitive cycling once the trace is visibly stable − after ≈ 2 seconds ) subsequently the above described cleaning procedure was operated continuously for 30 minutes . the following i / v curve was then again recorded : ( 5 single consecutive sweeps recorded + a sweep recorded after repetitive cycling once the trace is visibly stable − after ≈ 2 seconds ) the results are presented in fig1 a and 11b which respectively show the curves obtained after the 30 seconds initial cleaning and after the 30 minutes cleaning . for the purposes of comparison , fig1 shows an i / v curve obtained under the same conditions as those described above for a platinum electrode ( more specifically an electrode assembly of the type produced in accordance with example 3 above but without deposition of mercury ). fig1 shows an i / v curve obtained using the conditions described above for a hanging mercury drop electrode normalised by surface area . referring firstly to fig1 , it will be seen that the i / v curve for the hmde displays a characteristic “ water hump ” ( see left hand part of the curve illustrated in fig1 ). this “ water hump ” is not seen in the i / v curve for platinum illustrated in fig1 . referring now to fig1 a and 11b , it will firstly be noted that both curves display the characteristic “ water hump ” displayed by mercury ( cf fig1 ). thus the electrode assembly produced in accordance with example 3 displays the characteristics of a mercury electrode rather than a platinum electrode . moreover a comparison of fig1 a and 11b which are respectively before and after the 30 minute cleaning period demonstrates that the curves are identical indicating there is no significant loss in surface area and the surface character remains the same . the surface area of the electrode can be calculated accurately in accordance with equation ( 1 ) from the capacitance current using the value of specific capacitance for mercury measured for the water hump at ˜− 0 . 3v as ˜ 40 μf cm − 2 [ 27 ]). for the pe / hg composite electrode used in the above experiments this yielded a value for surface area of the electrode as ˜ 0 . 744 mm 2 . the surface area of the platinum disc electrode ( prior to mercury deposition ) was ˜ 0 . 724 mm 2 measured using an optical microscope . the calculated value for the composite electrode ( i . e . mercury deposited on the platinum ) was slightly higher than that for the flat disc providing a reasonable result for a flattened hemisphere because it lies between the bounds of a perfectly flat film and a hemisphere . values for surface area calculated in this fashion were used to produce the specific capacitance plots for composite electrodes coated with phospholipid monolayers in subsequent examples . this example demonstrates the ability of an electrode assembly produced in accordance with the procedure of example 3 to support phospholipid monolayers . the following 5 phospholipids were selected and varied only in the chemical functionality of their head group region : ( a ) 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine ( dopc ) ( b ) 1 , 2 dioleoyl - sn - glycero - 3 - phosphoethanolamine ( dope ) ( c ) 1 , 2 - dioleoyl - sn - glycero - 3 -[ phospho - rac -( 1 - glycerol )] ( sodium salt ) ( dopg ) ( d ) 1 , 2 - dioleoyl - sn - glycero - 3 -[ phospho - l - serine ] ( sodium salt ) ( dops ) ( e ) 1 , 2 - dioleoyl - sn - glycero - 3 - phospho ( ethylene glycol ) ( sodium salt ) ( dopeg ) the chemical structural formulae of the phospholipids ( a )-( e ) are shown in fig1 . solutions of each lipid of weight concentration 2 mg · ml − 1 dissolved in 1 : 4 chloroform : pentane were prepared separately yielding molar concentrations in the range 2 . 5 mmol · dm − 3 to 3 mmol · dm − 3 . the electrochemical cell was set - up as described in example 4 , containing 0 . 1 mol · dm − 3 kcl phosphate buffered at a concentration of 0 . 01 mol · dm − 3 to ph 7 . 4 and deaerated with argon for 30 minutes prior to introduction of the working electrode . 12 . 5 μl of each lipid solution was added by syringe to the electrolyte and the working electrode was then lowered through the solution / argon interface . the electrode was cleaned for 30 seconds using the in - situ electrochemical cleaning method described in example 4 before lifting the electrode through the solution interface briefly and re - submerging it to form an evenly coated layer . no discernible differences were observed in repetitive scans at 40 v · s − 1 over the potential range − 0 . 2 v to − 1 . 2 v for the electrode coated at open circuit , with the potential held at a constant − 0 . 2 v or while repetitively cycled over the above range ( data not shown ). cathodic scans of cyclic voltamograms recorded at 40 v s − 1 were converted to specific capacitance plots for the phospholipids ( a ) dopc , ( b ) dope , ( c ) dopg , ( d ) dops and ( e ) dopeg . the results are plotted in fig1 a - e respectively for which the thick line is the specific capacitance plot and the thin line is for an electrochemically cleaned electrode assembly of the type produced in example 3 ( i . e ., no deposited phospholipid ). it will be seen from the data presented in fig1 that the phospholipids act as variable dielectrics over the potential range between the potential of zero charge ( p . z . c .) for mercury and the layer &# 39 ; s desorption potential . the phospholipids impart selective chemical functionality to the surface and greatly affect the surface potential and capacitance profile producing unique finger print peaks relating to complex phase transitions of the absorbed layers . using the procedure of example 5 , the mercury surface of electrode assemblies produced in accordance with example 4 were separately coated with the phospholipids dopc , dops and dopg . for the dopc coated electrode , a cyclic voltammogram was recorded at 40 v · s − 1 over the potential range − 0 . 2 v to − 1 . 2 v . the electrode was then electrochemically cleaned in - situ for 30 seconds using the cleaning technique described in example 4 and the procedure repeated six times . the voltammograms were converted to specific capacitance plots and the average trace plotted . the results are shown in fig1 a in which the narrow error bars ( error bars =± 1 sd ) give a good indication as to the high level of measurement reproducibility between coatings . thus the phospholipid monolayer deposition exhibited a high degree of similarity between experiments . for the dops coated electrode , an initial scan was recorded at 40 v · s − 1 of the potential range − 0 . 2v to − 1 . 2v . the scan was repeated and recorded every 1 minute time interval for 30 consecutive minutes . the cyclic voltammograms were converted to specific capacitance plots and the average trace plotted . the results are shown in fig1 b in which the narrow error bars (± 1 sd ) indicate that the capacitance minimum and current peak remain stable over the course of the experiment thus demonstrating the monolayer integrity similarly remains stable and reproducible . to test stability and reproducibility in the case of continually cycling the potential between measurements , the dopg coated electrode was continuously cycled at 40 v · s − 1 over the potential range − 0 . 2 v to − 1 . 2 v resulting in interrogation 15 times per second , the cycle lasting 50 ms for the ramp + 40 ms software forced delay . the scans were recorded initially and after 5 , 15 and 25 minutes . the cyclic voltammograms were converted to specific capacitance plots and the traces overlaid to see any significant changes in the capacitance of the monolayer . the results are shown in fig1 c from which it can be seen that the traces overlay almost exactly indicating that monolayer integrity is sufficiently stable over the time period measured . this example demonstrates use of a prototype flow cell 400 of the type illustrated in fig1 which was constructed to establish “ proof - of - principle ” for use of an electrode assembly in accordance with the invention in a flow cell . the flow cell had an overall length of 5 cm , a height of 3 cm ( each of lower and upper portions 401 and 402 having a height of 1 . 5 cm ) and a width of 2 cm . injection port 406 as well as entry and exit channels 404 and 405 were of 4 mm diameter . bores 407 and 408 had a diameter of 2 mm and were angled at 45 degrees to exit channel 405 . the electrode assembly 300 was produced in accordance with the procedure of example 3 but passing 2c of charge ( rather than 1 c ). electrode assembly 300 had a length of 1 cm , a width of 5 mm and a depth of 0 . 5 mm . a ag / agcl microelectrode was provided in bore 407 and platinum counter electrode in bore 408 . contact pads 304 and 305 on the electrode assembly were individually connected to a potentionstat and could be addressed individually by means of a two way switch . lipid vesicle deposition dispersions of dopc and dops were prepared by dissolving 25 mg of powdered pure phospholipid in 50 : 50 chloroform / methanol and rotary evaporating in a glass round bottomed flask at 25 ° c . under light vacuum until dry . the residue was then re - suspended in 12 . 5 ml of phosphate buffered saline ( 0 . 1 mol · dm − 3 kcl , ph 7 . 4 ) to produce a 2 mg · ml − 1 dispersion which was then tip sonicated for 20 minutes to produce vesicles . the lipid layers were deposited by injecting ˜ 100 μl of 2 mg · ml − 1 dopc or dops vesicle dispersions into the injection port 406 upstream of the electrodes while electrolyte ( 0 . 1 m kcl phosphate buffered ( 10 mm ) to ph 7 . 4 ) composition , flow rate ?) was passed into and along inlet channel 404 , through measurement cell 403 and out through channel 405 at a rate of 5 ml per minute . it was found that the dopc layers could be deposited using the same conditions as adopted for the cleaning procedure described in example 4 but applied for ca 2 seconds . the potential cycling was stopped by opening the circuit when over - covered layer thinned sufficiently to exhibit the sharp phase transition peaks shown in fig1 which is rapid cyclic voltammogram at 36 v s − 1 of the electrode coated with dopc ( thick line ) measured with the experimental flow cell . for the purposes of comparison , fig1 also incorporates the corresponding trace for the pt / hg electrode ( thin line ) in the absence of lipid . the dops layers were deposited under different potential conditions from dopc due to the dops layers spreading rapidly at potentials & lt ;− 1 . 4 v . initial trials suggest that dops can be deposited over a lower potential range sweep with a cathodic apex of − 1 . 1 v . the dops layers were allowed slowly to build with time and successive additions to produce an i / v curve ( thick line ) as seen in fig1 which is a rapid cyclic voltammogram ( thick line ) at 38 v s − 1 of the electrode coated with dops measured within the experimental flow cell . for the purposes of comparison , fig1 also incorporates the trace ( thin line ) for the rapid cyclic voltammogram at 36 v s − 1 of the pt / hg electrode without lipid . both lipid layers ( dopc and dops ) on the electrodes proved to be stable over a period of & gt ; 10 minutes with electrolyte flowing at ˜ 4 . 5 ml · min − 1 and each could be deposited with a reasonable degree of reproducibility after cleaning the electrode in - situ and repeating the procedure . a feature of the prototype flow cell was the instability of the micro - reference electrode potential which was measured as + 260 mv when compared with the ag / agcl ( 3 . 5 mol · dm − 3 ) reference electrode used in the static cell . thus all potentials quoted from data within the flow cell are vs . a drifting ag / agcl reference . the exact drift can be evaluated by comparing the positions of the first or second phase transition peaks of dopc which occur at defined potentials on the hg surface . from fig1 and 18 a clear slant to the traces can be observed as well as broadening of the current peaks . this can be attributed to three contributing factors . firstly the cell was found not to be water tight due to an imperfect seal with the o - ring . ( any small electrolyte leaks can produce interfering faradaic currents when it comes into contact with the crocodile clips and contact pads .) secondly , the electrolyte contains a larger quantity of dissolved oxygen than the static cell which is more efficient at de - aeration . thirdly , the distance of the reference electrode from the working electrodes is slightly greater and there is a higher solution resistance in the flow cell due to the smaller reference electrode fritt that may become more easily blocked by phospholipid flows , the greater solution resistance influences the potential applied to the working electrodes through the phenomena of ohmic drop . in spite of the “ deficiencies ” of the prototype flow cell , this example clearly demonstrates a number of significant points . firstly , the mercury layers are stable in the electrolyte flow . secondly the phospholipids can be deposited on to the mercury layers and ( when interrogated by cyclic voltammetry ) give peaks corresponding with those obtained in a static cell , allowing for the “ deficiencies ” of the prototype flow cell . thirdly the lipid layers are stable in the electrolyte flow . fourthly the lipid layers can conveniently be deposited from the electrolyte flow by applying the appropriate potential to the electrode assemblies 301 and 302 of the electrode assembly 300 . this example demonstrates similarity in properties of a hanging mercury drop electrode ( hmde ) and pt / hg electrode as produced by the procedure of example 3 . the hmde was based on using a capillary with a diameter of 0 . 1 mm so as to provide a surface area for the mercury drop about the same as the surface area of the mercury in the electrode in accordance with the invention . the hmde and the electrode of the invention were compared side - by - side in a static cell configuration . all experiments were carried out in 0 . 1 m potassium chloride solution . measurements were taken at 75 hz between − 0 . 4 to − 1 . 15 v with 4 . 95 mv rms at a scan rate of 5 mv capacitance — potential scans for the hmde and the composite pt / hg electrode are shown in fig1 a and b respectively . although there is a slight difference in the capacitance values shown in fig1 a and b , the shape of the plots indicates close similarity in the properties of the two electrodes . this example compares a hmde electrode with an electrode as produced in accordance with example 3 , both coated with a monolayer film of 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine ( dopc ), new layers of which were deposited between consecutive scans . experiments were performed in 0 . 1m kcl . a negative going ‘ forward ’ potential scan was performed between − 0 . 4 v and − 1 . 15 v , and then followed immediately by a positive going ‘ reverse ’ scan from − 1 . 15 v to − 0 . 4 v with 4 . 95 mv rms at a scan rate of 5 mvs − 1 . the results for the hmde are shown in fig2 a and b , and for the electrode of the invention are shown in fig2 a and b respectively . as with the scans of the “ bare ” electrodes in the electrolyte solution ( results shown in fig1 ), both electrodes show comparable results . the ‘ reverse ’ potential scans show that the electrode of the invention is slightly more stable than the hmde electrode . the procedure of example 9 was repeated but with the incorporation in the electrolyte of chlorpromazine at a concentration of 0 . 5 μmol dm − 3 . the structure of chlorpromazine is as shown below : the results for hmde are represented by the dark lines in fig2 a and b and for the electrode of the invention are represented by the dark lines in fig2 c and d respectively . for the purposes of comparison , these figures also incorporate the results obtained in example 9 . the procedure of example 9 was repeated but with the incorporation in the electrolyte of promethazine at a concentration of 0 . 5 μmol dm − 3 . the structure of promethazine is shown below : the results for hmde are represented by the dark lines in fig2 a and 22b and for the electrode of the invention are represented by the dark lines in fig2 c and d respectively . for the purposes of comparison , these figures also incorporate the results obtained in example 9 . in this case , both electrodes show response to the test compound but with the electrode of the invention displaying the stronger response ( greater depression of the peaks ) than the hmde . the procedure of example 9 was repeated but with the incorporation in the electrolyte of h16 at a concentration of 0 . 5 μmol dm − 3 . the structure of h16 is shown below . the results for hmde are represented by the dark lines in fig2 a and 23b and the electrode of the invention are represented by the dark lines in fig2 c and d respectively . for the purposes of comparison , these figures also incorporate the results obtained in example 9 . in this case , the electrode of the invention shows a far greater response than that seen with the hmde , thus indicating greater sensitivity of the former than the latter . example 12 was repeated but using 5 μmol dm − 3 of h16 . the results for hmde are represented by the dark lines in fig2 a and b and the electrode of the invention are represented by the dark lines in fig2 c and d respectively . for the purposes of comparison , these figures also incorporate the results obtained in example 9 . fig2 a and b demonstrate a large response to the test compound that is easily visible on both electrodes , with the electrode of the invention still showing the slightly larger response . following the procedure of example 9 , pt / hg composite electrodes produced in accordance with the procedure of example 3 and provided individually with monolayers of dopc , dope and dops were evaluated . the results are shown in fig2 a - c which show the results for dopc , dope and dops respectively ( note the difference in vertical scale for dops compared to the other two lipids shown ). the results show that monolayers of all three types of phospholipid can be formed on the electrodes and that each produces a different characteristic trace in the ac voltammetry experiments . the procedure of example 14 was repeated but incorporating 0 . 5 mol dm − 3 chlorpromazine in the electrolyte solution . the results were illustrated by the dark lines in fig2 a - c which show the results for dopc , dope and dops respectively . for the purposes of comparison , fig2 a - c also incorporate the results shown in fig2 . fig2 clearly demonstrates interaction of the chlorpromazine with the lipid monolayers ( cf the superimposed results from fig2 for the “ non - interacting ” monolayers ). the procedure of example 10 was repeated for electrodes produced in accordance with example 3 and incorporating a dopc monolayer to evaluate the system for the following compounds all provided in the electrolyte at a concentration of 0 . 5 mol dm − 3 , save for ( f ) limonene which was used at a concentration of 5 mol dm − 3 : the results are represented by the dark lines shown in fig2 a - 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