EP2545366A1 - Biosensor apparatus and use thereof - Google Patents

Biosensor apparatus and use thereof

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Publication number
EP2545366A1
EP2545366A1 EP11719034A EP11719034A EP2545366A1 EP 2545366 A1 EP2545366 A1 EP 2545366A1 EP 11719034 A EP11719034 A EP 11719034A EP 11719034 A EP11719034 A EP 11719034A EP 2545366 A1 EP2545366 A1 EP 2545366A1
Authority
EP
European Patent Office
Prior art keywords
sample
electrolyte
inlet
phospholipid
mercury
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP11719034A
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German (de)
French (fr)
Inventor
Andrew Lawrence Nelson
Alexander Vakourov
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University of Leeds
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University of Leeds
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Filing date
Publication date
Application filed by University of Leeds filed Critical University of Leeds
Publication of EP2545366A1 publication Critical patent/EP2545366A1/en
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
    • G01N2030/8854—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds involving hydrocarbons

Definitions

  • the present invention relates to a biosensor apparatus incorporating an electrode assembly having an electrode intended to incorporate a phospholipid layer simulating a biomembrane and to the use of the biosensor apparatus.
  • the biosensor apparatus may be used, for example for electrochemically analysing a sample to determine the presence (or otherwise) of a species having biomembrane activity (e.g. a toxin) or for investigating whether a species (e.g. a potential pharmaceutical has biomembrane activity).
  • the present invention relates to a development of the biosensor described more fully in our earlier PCT application published as WO 2009/016366.
  • the aforementioned prior PCT specification discloses an electrode assembly comprising at least one working electrode comprised of a conductive carrier substrate (e.g. platinum) having a surface coated with mercury immobilised on the surface of the substrate. Additionally the surface of the mercury remote from the substrate is coated with a phospholipid layer.
  • the biosensor apparatus further includes a counter electrode and a reference electrode. Measurements to investigate biomembrane activity of a species in a carrier liquid may be made by monitoring the phospholipid layer on the mercury electrode by rapid cyclic voltammetry using a saw tooth waveform at a rapid ramp rate.
  • the resulting current is proportional to the capacitance of the surface of the electrode.
  • the capacitance shows sharp peaks, representing phase changes of the phospholipid layer which correspond to its fluidity and (given that the electrolyte liquid does not contain any biomembrane active components) is very characteristic of the pure phospholipid.
  • the presence of a species have biomembrane activity changes the fluidity and affects the phase changes, thus influencing the form of the peaks.
  • Fig 10 of the aforementioned PCT application discloses a flow cell arrangement for use in effecting measurement of the biomembrane activity.
  • a single port (referenced as 406) through which (i) phospholipid to be deposited on the mercury electrode, and (ii) sample to be monitored are separately introduced at various stages of the measurement procedure.
  • a biosensor apparatus for analysing a sample comprising:
  • a biosensor unit having a liquid flow path therethrough between an inlet and an outlet of the unit, said unit comprising
  • an electrode assembly comprising at least one working electrode comprised of a conductive carrier substrate having a surface coated with mercury immobilised on the surface of the substrate,
  • (v) means for determining variations in the differential capacitance of phospholipid deposited on the mercury as a function of potential against the counter electrode.
  • a method of analysing a sample to determine biomembrane activity (or otherwise) of the sample using the apparatus of the first aspect of the present invention comprising the steps of: (i) supplying the inlet of the apparatus a flow of electrolyte containing a phospholipid and effecting deposition of the phospholipid on the surface of the mercury remote from the carrier substrate,
  • the sample supply arrangement for introducing the sample to be analysed into electrolyte flow is adapted to introduce a metered amount of the sample and may, for example, be a syringe.
  • the apparatus is preferably such that, in the direction of electrolyte flow to the inlet of the biosensor unit, the phospholipid supply arrangement is downstream of the sample supply arrangement.
  • the liquid supply arrangement is adapted selectively to supply either control electrolyte or electrolyte containing sample to the inlet of the biosensor unit.
  • This enables separate flow injection systems for the sample and the phospholipid dispersion to be carried out.
  • the separation of the sample from the phospholipid dispersion facilitates the injection of sample (e.g. nanoparticle dispersions) with defined concentration and pH from buffer.
  • the liquid supply arrangement may be adapted to provide a first flow path for supplying a control electrolyte of the biosensor unit and a second flow path for supplying electrolyte containing sample to the inlet of the biosensor unit.
  • the first flow path may include a first reservoir for holding control electrolyte.
  • the second flow path may be adapted to operate as a continuous loop.
  • the second flow path may incorporate a second reservoir for holding electrolyte (preferably in a predetermined volume).
  • the sample supply arrangement may introduce the sample into the second reservoir.
  • Conductive substrates for use in the invention i.e. the substrate on which the mercury is deposited
  • Conductive substrates for use in the invention preferably have a resistivity of less than 1 ohm metre, more preferably less than 1 x 10 "2 ohm metre and ideally less than 1 x 10 "3 ohm metre.
  • the carrier substrate may be a metal selected from the group consisting of iridium, platinum, palladium and tantalum which are selected as "carriers" because of their refractory inert properties and their low solubility in mercury and because the mercury can be deposited on such metals (e.g. by electrodeposition) to give a uniform film.
  • the smooth mercury surface allows for defect-free formations of the phospholipid layer.
  • a further conductive carrier substrate that may be employed in the invention is carbon, e.g. in the form of graphite or glassy carbon. Platinum is the preferred carrier substrate.
  • platinum has a particularly appropriate solubility with respect to mercury to allow production of an "amalgam-like" joint which holds the mercury relatively strongly on the platinum whilst providing a good surface for the mercury to allow phospholipid deposition and provide good membrane activity.
  • the phospholipid to be provided as a layer on the surface of the mercury may be saturated or may have a degree of unsaturation.
  • suitable phospholipids include dimyristoyl, phosphatidyl choline (D PC - saturated), dioleoyi phosphhtidyl choline (DOPC - unsaturated) and egg lecithin (egg PC - saturated/unsaturated)
  • D PC dimyristoyl
  • DOPC - unsaturated dioleoyi phosphhtidyl choline
  • egg lecithin egg PC - saturated/unsaturated
  • the head group may be amine based as in dioleoyi phosphatidylethanolamine (DOPE) or hydroxyl based as in 1 ,2-dioleoyl-sn-glycero-3-phospho(ethylene glycol) (sodium salt) or contain a combination of chemical functional groups as in 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-(succinyl) (sodium salt). These functional groups may be present in 'natural' lipids or may be synthesized to confer a desired chemical surface.
  • DOPE dioleoyi phosphatidylethanolamine
  • contain a combination of chemical functional groups as in 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-(succiny
  • Each lipid present as a homogeneous monolayer or as a fraction of a heterogeneous monolayer will confer unique properties to that layer that may be observed through the layers capacitance over the interrogation potential window.
  • Modification of the phospholipid head group may be used and be important in the sensor's operation due to this being the functionalised surface that is presented directly to the electrolyte.
  • the layers properties may be tuned by changing the length of the carbon chains and their saturation or through the modification of the head groups.
  • phospholipids with two 9-cis-octadecenoic chains are fluid at room temperature and capable of forming impermeable monolayers on mercury. They exhibit sharp pseudo capacitative phase transitions within the potential range interrogated by rapid cyclic voltammetry.
  • dimyristoyl lipids (incorporating d 4 saturated chains) have the advantage of being less susceptible to oxidation than dioleoyl lipids but exhibit less prominent phase transitions.
  • the phospholipid layer may be associated with additional components for modifying the properties of that layer. These components may be incorporated within the layer or covalently tethered thereto and examples include peptides (to form ion channels), oligonucleotides or molecules complementary to the target molecule.
  • the phospholipid layer may be cleaned off the mercury coating and a fresh layer applied.
  • the successively deposited phospholipid layers provide a high degree of reproducibility in terms of the results obtained by interrogating the layers by cyclic voltammetry.
  • the cleaning of the phospholipid from the mercury surface may be effected by scanning the working electrode in a cathodic direction (e.g. over the range (-0.2 V to -2.625 V) @ 97 Vs "1 ) with the electrode being immersed in electrolyte so as to desorb any contaminating organic material into the bulk solution.
  • a cathodic direction e.g. over the range (-0.2 V to -2.625 V) @ 97 Vs "1
  • similar scan conditions when applied for much shortly periods of time than used for cleaning can be used to deposit the phospholipid layer on the mercury film.
  • the electrode will be immersed in electrolyte which incorporates the phospholipid to be deposited.
  • the phospholipid may be added to the aqueous electrolyte in the form of a dispersion prepared by agitation (e.g. using sonication) of the phospholipid in an aqueous medium.
  • the mercury electrode may be monitored by cyclic voltammetry and deposition of the layer may be detected by the appearance in a cyclic voltammogram of a trace characteristic of the phospholipid. With the appearance of this trace, the deposition procedure will be complete.
  • the mercury surface of the composite electrode i.e. the electrode comprised of the conductive substrate and the mercury coating
  • the biosensor functions by monitoring the lipid layer and in particular the modification thereof due to the presence in a sample under investigation of a species having biomembrane activity. Measurements are made by voltammetry to determine variations in the differential capacitance of the phospholipid as a function of voltage against the reference electrode, in a similar manner to that disclosed in GB-B 2 193 326. Most preferably measurement is by means of rapid cyclic voltammetry, preferably using a sawtooth waveform with a ramp rate of ⁇ 1 Vs "1 (e.g. 40-100 Vs "1 ). The voltage excursion used in rapid cyclic voltammetry may be from -0.4 V to -1.2 V vs Ag/AgCI 3.5 M KCI.
  • the output current (i) is proportional to the differential capacitance (C d ) as indicated by the equation:
  • the experimental set-up for RCV involves the application of the sawtooth wave form using a function generator with input to a potentiostat which applies the waveform to the working electrode.
  • the resulting current response is recorded via an acquisition board and plotted against the applied waveform.
  • DC cyclic voltammetry provides rapid assessment of the layer's capacity over a defined potential window specific to the phospholipid monolayer's structure and environment.
  • measurement may be made by ac voltammetry using, for example, a voltage ramp of about 5 mV s " with a superimposed sinusoidal voltage of frequency, f, about 75 Hertz and of amplitude, ⁇ , about 0.005 V.
  • the output ac current is separated into both in phase and out of phase components.
  • the out of phase current (i") is proportional to the differential capacitance (C d ) as expressed by the equation:
  • the experimental set-up for ac voltammetry involves adding the sinusoidal waveform to the above voltage ramp and inputting to a potentiostat which applies the resulting waveform to the working electrode.
  • the ac current response is fed into a lock-in amplifier where the in phase and out of phase components with the applied ac waveform of the current are separated and recorded on a data acquisition system.
  • the out of phase current is plotted against the ramp voltage.
  • the working electrode may for example be circular and/or have a maximum surface dimension in any direction of 2pm to 1 mm, although dimensions outside this range are not precluded.
  • the working electrode may be a microelectrode and preferably sized such that the mercury has a maximum surface dimensions of 2pm to 10pm in any direction.
  • the or each working electrode may, for example, be circular with a diameter of 2pm to 10pm. Such dimensions serve to maximise the edge-to area and thereby enhance stability of the phospholipid layer.
  • the mercury layer may be formed by eletrodeposition.
  • the amount of mercury deposited should be sufficient to give a continuous film of the mercury on the conductive carrier substrate.
  • Increasing the thickness of the mercury layer will enhance the stability of that layer and allow repeated the mercury layer to allow repeated cycles of phospholipid layer deposition, sample measurement and removal of the phospholipid layer so that one electrode may be used many times without disruption of the mercury.
  • thickness of mercury layer will be a consideration for use of the electrode assembly in a flow cell where the mercury layer is required to withstand forces associated with liquid flows through the cell.
  • the amount of charge required for the electrodeposition process will depend on the required thickness of the mercury layer and this will in turn depend on factors such as the surface area of the conductive substrate (on to which the mercury is to be electrodeposited) and the concentration of mercury ions in the mercury deposition electrolyte.
  • Controlled electrodeposition of mercury from a solution containing Hg 2+ ions may be effected within an electrochemical cell.
  • the mercury deposition electrolyte may contain HCIO4 which prevents oxidation of the Hg species. However the lipid interrogation will not take place in this acidic solution and will instead be in a more benign electrolyte such as 0.1 M KCI.
  • electrodeposition of mercury can be effected by pipetting a drop of the base electrolyte (e.g. HCIO 4 + Hg(ll)) onto the surface of the first insulator.
  • the electrodeposition may be facilitated by applying a potential of —0.4V vs. 3.5 M KCI Ag/AgCI to the working electrode surface in the deposition solution and recording the charge passed which is directly proportional to the mass of mercury deposited the deposition can be stopped by breaking the circuit. This allows precise control over the amount of Hg deposited.
  • the assembly can be transferred to a neutral electrolyte such as 0.1 M KCI and the electrode surface eiectrochemicaliy cleaned by applying an extreme negative potential ⁇ -2V evolving hydrogen gas to 'scrub' the surface free from organics.
  • a neutral electrolyte such as 0.1 M KCI and the electrode surface eiectrochemicaliy cleaned by applying an extreme negative potential ⁇ -2V evolving hydrogen gas to 'scrub' the surface free from organics.
  • the biosensor of the invention may be used, for example, for determining (i) the presence or otherwise in a sample of a species known to have biomembrane activity or (ii) whether or not a particular substance has biomembrane activity.
  • the measurements may be made by voltammetry, (e.g. rapid cyclic voltammetry or ac- voltammetry effected in conventional manner such as along the lines disclosed in GB- A-2 193 326 (see for example Fig 1 thereof).
  • RCV will be used for rapid interrogation of the sensor surface and in particular to determine the effect of the interaction of the two capacitance of the phospolipid which are particular sensitive to interactions.
  • the invention has applications in a wide number of fields. These include:
  • detection of toxic gases and explosive vapours which (provided they interact with the monolayer surface) may be diagnosed using a multivariate analysis approach depending on the strength of the interaction with monolayers of varying chemical functionality;
  • biomembrane active compounds such as (i) toxic biomembrane active peptides produced by blooms of cyanobacteria and dinoflagelates or (ii) pollutants.
  • the system may be calibrated for sensor application with a series of compounds eg polycyclic aromatic hydrocarbons, phenothiazine drugs and anti-microbial peptides which are known to modify the structure of phospholipid layers on electrodes.
  • a series of compounds eg polycyclic aromatic hydrocarbons, phenothiazine drugs and anti-microbial peptides which are known to modify the structure of phospholipid layers on electrodes.
  • the disruption/modification of the phospholipid layer has been well characterised electrochemically.
  • the present invention is particularly useful for analysing samples containing nanoparticles but may be used with other types of samples, e.g. as fully disclosed in the WO specification.
  • Figs 1 (a)-(i) illustrate one embodiment of biosensor apparatus in accordance with the invention in successive stages of operation
  • Figs 2 (a) and (b) show the results of the Example detailed below.
  • the illustrated apparatus comprises a biosensor unit 1 (in the form of a flow cell) incorporating a bionsensor 2 of the type described more fully in WO 2009/016366 and comprising mercury immobilised on a conductive substrate platinum although other conductive substrate as disclosed in WO 2009/016366 may be employed.
  • Biosensor unit 1 has a liquid inlet 3 and liquid outlet 4 associated with a single channel peristalitic pump 5 (available from Cole Palmer Instrument) located upstream of inlet 3 and control valves 6 and 7 which regulate supply and discharge of liquid to and from biosensor unit 1 along various flow paths in the manner described more fully below.
  • Valves 6 and 7 are universal valve switching modules with six channels.
  • a syringe 8 located immediately upstream of the inlet 3 of the flow cell 1 and arranged to discharge its contents into the flow path leading to the inlet 3.
  • Syringe 8 contains a phospholipid dispersion, the phospholipds being for the purpose of forming a monolayer on the mercury surface of the electrode in the biosensor 2 in the manner described more fully in WO 2009/016366.
  • a further syringe 9 is provided holding a sample to be analysed and is arranged to discharge a known (metered) amount of this sample into a reservoir 10 containing a known volume of buffered electrolyte.
  • Control electrolyte (buffered) is provided in a further reservoir 11.
  • Argon is fed to reservoirs 10 and 1 1 as depicted by the labelled arrows.
  • biosensor 2 is of the general type described in WO 2009/016366 and, in the present case, comprises the working electrode (i.e. mercury deposited on conductive substrate) and a counter electrode.
  • a separate Ag/AgCI reference electrode 12 is provided for the biosensor unit 1 , all three electrodes (working, counter and reference) being connected to a potentiostat 13 controlled by AUTOLAB software and associated with a monitor 14 for displaying results.
  • Bionsensor unit 1 may be of a similar device to the flow cell illustrated in Fig 10 of WO 2009/016366 save that inlet port 406 of that flow cell is not required.
  • Fig 1 (a) shows the apparatus in its "non-operational" condition in which there is no liquid circulation through the biosensor unit 1. In this "non-operational" condition reservoirs 10 and 1 1 are both charged with electrolyte, syringe 8 is charged with phospholipid dispersion and syringe 9 is charged with sample to be analysed.
  • valves 6 and 7 are set so that control electrolyte from reservoir 1 1 passes in sequence through valve 7, pump 5, biosensor unit 1 , valve 7, valve 6 and then to discharge, all as represented by the arrows shown in Fig 1 (b).
  • a trace may be displayed on monitor 14 showing the signal obtained from the bare mercury electrode (see Fig 1 (c)).
  • the plunger of syringe 8 may now be depressed to inject phospholipid dispersion into the flow line between pump 5 and inlet 3 of the biosensor unit 1 (Fig 1 (d)).
  • a monolayer of phospholipid may be deposited on the mercury surface of the working electrode of biosensor 2. These conditions may involve using rapid cyclic voltammetry (RCV) employing an "up-and-down" voltage ramp between -0.2 and -3 V. Successful phospholipid monolayer formation on the mercury electrode may be checked using rapid RCV between -0.2 and -1.8 V and checking the standard current-potential profile (see screen trace depicted on monitor 14 in Fig 1 (d)).
  • RCV rapid cyclic voltammetry
  • valves 6 and 7 are set so that electrolyte from reservoir 10 runs round a continuous flow path whereby this electrolyte from reservoir 10 is passed in sequence through valve 7, pump 5, bionsensor unit 1 , back to valve 7, to valve 6 and then returned to reservoir 10 (see arrows in Fig 1 (e)).
  • the plunger of syringe 9 is depressed (Fig 1 (f)) to discharge a known volume of sample into reservoir 10, the sample then circulating around the continuous flow path as described for Fig 1 (e).
  • valve 6 is set (Fig 1(g)) so that the electrolyte (containing sample) previously circulating back to reservoir 10 is now discharged to waste as depicted by the arrows in Fig 1(g).
  • valves 6 and 7 are set (Fig 1 (h)) so that electrolyte from reservoir 11 passes through the apparatus as depicted by the arrows and is then discharged.
  • the mercury electrode may be "cleaned" by removal of the phospholipid using techniques as disclosed in WO 2009/016366. This may involve scanning the working electrode in a cathodic direction, e.g. over the range -0.2 V to -2.625 V @ 97 Vs "1 with the electrode being immersed in electrolyte so as to desorb any contaminating organic material into the bulk solution.
  • valve 6 is set (Fig 1 (i)) in readiness for a further cycle of the apparatus.
  • a significant advantage of the illustrated apparatus is that the separation of the sample (contained in syringe 8) from the phospholipid dispersion (contained in syringe 9) facilitates the injection of sample in electrolyte as sample with defined concentration and pH from buffer.
  • the illustrated apparatus is suitable particularly, but not exclusively, for measuring the effect of nanoparticle dispersions on the phospholipid layer.
  • the invention is further illustrated by the following non-limiting Example.
  • Apparatus as illustrated in Fig 1 was used to determine the effect of LUDOX® Si0 2 nanoparticles of different particle sizes on a phospholipid layer deposited on the mercury electrode.
  • SM30 with an Si0 2 concentration of 2.92 m
  • TM50 with a SI0 2 concentration of 3.3 mM
  • the carrier electrolyte in reservoir 1 1 was 0.1 mol dm "3 KCI buffered with borate 0.1 mol dm '3 /phosphate 0.25 mol dm "3 /tris 0.1 mol dm "3 buffers at pH 10.7.
  • the same electrolyte was provided for reservoir 10. All samples were purified and redispersed in buffer prior to the tests.
  • Figures 2(a) and (b) display the results for nanoparticle biomembrane activity as a function of time in respect of the SM30 (0 and 4 min) and TM50 (0 and 6 min) respectively.
  • the x axis label in Fig 2 is l/microamps.
  • the nanoparticle activity is displayed as a depression of the two current peaks on the displayed RCV plot on the monitor. The activity is shown to depend closely on the nanoparticle dispersion tested.

Abstract

A biosensor apparatus for analysing a sample comprises: (i) a biosensor unit (1) having a liquid flow path therethrough between an inlet (3) and an outlet (4) of the unit, said unit comprising (a) an electrode assembly comprising at least one working electrode comprised of a conductive carrier substrate having a surface coated with mercury immobilised on the surface of the substrate, (b) a counter electrode, and (c) a reference electrode (12), (ii) a liquid supply arrangement (11 ) for supplying a flow of electrolyte to the inlet (3) of the biosensor unit, (iii) a phospholipid supply arrangement (8) for introducing phospholipid into electrolyte flow to the inlet (3) of the biosensor unit for deposition on the exposed surface of the mercury, (iv) a sample supply arrangement (9) for introducing the sample to be analysed into electrolyte flow to the inlet (3) of the biosensor unit, and (v) means (13) for determining variations in the differential capacitance of phospholipid deposited on the mercury as a function of potential against the counter electrode.

Description

BIOSENSOR APPARATUS AND USE THEREOF
The present invention relates to a biosensor apparatus incorporating an electrode assembly having an electrode intended to incorporate a phospholipid layer simulating a biomembrane and to the use of the biosensor apparatus. The biosensor apparatus may be used, for example for electrochemically analysing a sample to determine the presence (or otherwise) of a species having biomembrane activity (e.g. a toxin) or for investigating whether a species (e.g. a potential pharmaceutical has biomembrane activity).
The present invention relates to a development of the biosensor described more fully in our earlier PCT application published as WO 2009/016366. Briefly, the aforementioned prior PCT specification discloses an electrode assembly comprising at least one working electrode comprised of a conductive carrier substrate (e.g. platinum) having a surface coated with mercury immobilised on the surface of the substrate. Additionally the surface of the mercury remote from the substrate is coated with a phospholipid layer. The biosensor apparatus further includes a counter electrode and a reference electrode. Measurements to investigate biomembrane activity of a species in a carrier liquid may be made by monitoring the phospholipid layer on the mercury electrode by rapid cyclic voltammetry using a saw tooth waveform at a rapid ramp rate. At low voltages, the resulting current is proportional to the capacitance of the surface of the electrode. At high voltages, the capacitance shows sharp peaks, representing phase changes of the phospholipid layer which correspond to its fluidity and (given that the electrolyte liquid does not contain any biomembrane active components) is very characteristic of the pure phospholipid. The presence of a species have biomembrane activity changes the fluidity and affects the phase changes, thus influencing the form of the peaks.
Fig 10 of the aforementioned PCT application discloses a flow cell arrangement for use in effecting measurement of the biomembrane activity. In the arrangement of Fig 10 of the earlier PCT application, there is a single port (referenced as 406) through which (i) phospholipid to be deposited on the mercury electrode, and (ii) sample to be monitored are separately introduced at various stages of the measurement procedure. According to a first aspect of the present invention there is provided a biosensor apparatus for analysing a sample, the apparatus comprising:
(i) a biosensor unit having a liquid flow path therethrough between an inlet and an outlet of the unit, said unit comprising
(a) an electrode assembly comprising at least one working electrode comprised of a conductive carrier substrate having a surface coated with mercury immobilised on the surface of the substrate,
(b) a counter electrode, and
(c) a reference electrode,
(ii) a liquid supply arrangement for supplying a flow of electrolyte to the inlet of the biosensor unit,
(iii) a phospholipid supply arrangement for introducing phospholipid into electrolyte flow to the inlet of the biosensor unit for deposition on the exposed surface of the mercury,
(iv) a sample supply arrangement for introducing the sample to be analysed into electrolyte flow to the inlet of the biosensor unit, and
(v) means for determining variations in the differential capacitance of phospholipid deposited on the mercury as a function of potential against the counter electrode.
According to a second aspect of the present invention there is provided a method of analysing a sample to determine biomembrane activity (or otherwise) of the sample using the apparatus of the first aspect of the present invention, the method comprising the steps of: (i) supplying the inlet of the apparatus a flow of electrolyte containing a phospholipid and effecting deposition of the phospholipid on the surface of the mercury remote from the carrier substrate,
(ii) supplying to the inlet of the apparatus a flow of electrolyte containing the sample to be analysed, and
(iii) using a voltammetric technique to determine the biomembrane activity.
By providing, in the apparatus of the invention, separate phospholipid supply and sample supply arrangements considerable flexibility is introduced into the apparatus as compared to the apparatus described in WO 2009/016366. More particularly, it is possible to provide separate flow injection systems for the sample and the phospholipid. The separation of the sample supply arrangement from the phospholipid supply arrangement facilitates the injection of a sample with defined concentration and pH for monitoring in the biosensor unit.
Preferably the sample supply arrangement for introducing the sample to be analysed into electrolyte flow is adapted to introduce a metered amount of the sample and may, for example, be a syringe.
The apparatus is preferably such that, in the direction of electrolyte flow to the inlet of the biosensor unit, the phospholipid supply arrangement is downstream of the sample supply arrangement.
In particularly preferred embodiments of the invention, the liquid supply arrangement is adapted selectively to supply either control electrolyte or electrolyte containing sample to the inlet of the biosensor unit. This enables separate flow injection systems for the sample and the phospholipid dispersion to be carried out. The separation of the sample from the phospholipid dispersion facilitates the injection of sample (e.g. nanoparticle dispersions) with defined concentration and pH from buffer. The liquid supply arrangement may be adapted to provide a first flow path for supplying a control electrolyte of the biosensor unit and a second flow path for supplying electrolyte containing sample to the inlet of the biosensor unit. In such a case, the first flow path may include a first reservoir for holding control electrolyte. Alternatively or additionally the second flow path may be adapted to operate as a continuous loop. The second flow path may incorporate a second reservoir for holding electrolyte (preferably in a predetermined volume). The sample supply arrangement may introduce the sample into the second reservoir.
Conductive substrates for use in the invention (i.e. the substrate on which the mercury is deposited) preferably have a resistivity of less than 1 ohm metre, more preferably less than 1 x 10"2 ohm metre and ideally less than 1 x 10"3 ohm metre.
The carrier substrate may be a metal selected from the group consisting of iridium, platinum, palladium and tantalum which are selected as "carriers" because of their refractory inert properties and their low solubility in mercury and because the mercury can be deposited on such metals (e.g. by electrodeposition) to give a uniform film. The smooth mercury surface allows for defect-free formations of the phospholipid layer. A further conductive carrier substrate that may be employed in the invention is carbon, e.g. in the form of graphite or glassy carbon. Platinum is the preferred carrier substrate.
Without wishing to be bound by theory) we believe platinum has a particularly appropriate solubility with respect to mercury to allow production of an "amalgam-like" joint which holds the mercury relatively strongly on the platinum whilst providing a good surface for the mercury to allow phospholipid deposition and provide good membrane activity.
The phospholipid to be provided as a layer on the surface of the mercury may be saturated or may have a degree of unsaturation. Examples of suitable phospholipids include dimyristoyl, phosphatidyl choline (D PC - saturated), dioleoyi phosphhtidyl choline (DOPC - unsaturated) and egg lecithin (egg PC - saturated/unsaturated) Such lipids incorporate choline-based head groups and other lipids incorporating this head group may be used. However the head group may be amine based as in dioleoyi phosphatidylethanolamine (DOPE) or hydroxyl based as in 1 ,2-dioleoyl-sn-glycero-3-phospho(ethylene glycol) (sodium salt) or contain a combination of chemical functional groups as in 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-(succinyl) (sodium salt). These functional groups may be present in 'natural' lipids or may be synthesized to confer a desired chemical surface. Each lipid present as a homogeneous monolayer or as a fraction of a heterogeneous monolayer will confer unique properties to that layer that may be observed through the layers capacitance over the interrogation potential window. Modification of the phospholipid head group may be used and be important in the sensor's operation due to this being the functionalised surface that is presented directly to the electrolyte. The layers properties may be tuned by changing the length of the carbon chains and their saturation or through the modification of the head groups.
More specifically, phospholipids with two 9-cis-octadecenoic chains are fluid at room temperature and capable of forming impermeable monolayers on mercury. They exhibit sharp pseudo capacitative phase transitions within the potential range interrogated by rapid cyclic voltammetry. By comparison, dimyristoyl lipids (incorporating d4 saturated chains) have the advantage of being less susceptible to oxidation than dioleoyl lipids but exhibit less prominent phase transitions.
If desired the phospholipid layer may be associated with additional components for modifying the properties of that layer. These components may be incorporated within the layer or covalently tethered thereto and examples include peptides (to form ion channels), oligonucleotides or molecules complementary to the target molecule.
The phospholipid layer may be cleaned off the mercury coating and a fresh layer applied. We have established that (for a particular composite electrode comprised of the conducting substrate with mercury coating) the successively deposited phospholipid layers provide a high degree of reproducibility in terms of the results obtained by interrogating the layers by cyclic voltammetry.
Conveniently, the cleaning of the phospholipid from the mercury surface may be effected by scanning the working electrode in a cathodic direction (e.g. over the range (-0.2 V to -2.625 V) @ 97 Vs"1) with the electrode being immersed in electrolyte so as to desorb any contaminating organic material into the bulk solution. Surprisingly we have found that similar scan conditions when applied for much shortly periods of time than used for cleaning can be used to deposit the phospholipid layer on the mercury film. For the purposes of this deposition, the electrode will be immersed in electrolyte which incorporates the phospholipid to be deposited. The phospholipid may be added to the aqueous electrolyte in the form of a dispersion prepared by agitation (e.g. using sonication) of the phospholipid in an aqueous medium. During the deposition procedure, the mercury electrode may be monitored by cyclic voltammetry and deposition of the layer may be detected by the appearance in a cyclic voltammogram of a trace characteristic of the phospholipid. With the appearance of this trace, the deposition procedure will be complete.
The mercury surface of the composite electrode (i.e. the electrode comprised of the conductive substrate and the mercury coating) may be re-used in a repetitive cycle of steps (i) to (iii) below:
(i) depositing a fresh phospholipid layer;
(ii) effecting a measurement on a sample; and
(iii) cleaning the electrode to remove the "used " phospholipid.
Consequently there is no need to regenerate the mercury coating for each measurement.
The biosensor functions by monitoring the lipid layer and in particular the modification thereof due to the presence in a sample under investigation of a species having biomembrane activity. Measurements are made by voltammetry to determine variations in the differential capacitance of the phospholipid as a function of voltage against the reference electrode, in a similar manner to that disclosed in GB-B 2 193 326. Most preferably measurement is by means of rapid cyclic voltammetry, preferably using a sawtooth waveform with a ramp rate of≥ 1 Vs"1 (e.g. 40-100 Vs"1). The voltage excursion used in rapid cyclic voltammetry may be from -0.4 V to -1.2 V vs Ag/AgCI 3.5 M KCI. The output current (i) is proportional to the differential capacitance (Cd) as indicated by the equation:
Cd = i / (v x A) where A is the electrode area and v is the ramp rate.
The experimental set-up for RCV involves the application of the sawtooth wave form using a function generator with input to a potentiostat which applies the waveform to the working electrode. The resulting current response is recorded via an acquisition board and plotted against the applied waveform.
DC cyclic voltammetry provides rapid assessment of the layer's capacity over a defined potential window specific to the phospholipid monolayer's structure and environment.
Alternatively measurement may be made by ac voltammetry using, for example, a voltage ramp of about 5 mV s" with a superimposed sinusoidal voltage of frequency, f, about 75 Hertz and of amplitude, ΔΕ, about 0.005 V. The output ac current is separated into both in phase and out of phase components. The out of phase current (i") is proportional to the differential capacitance (Cd) as expressed by the equation:
Cd = i" / (2TT X f x ΔΕ x A)
The experimental set-up for ac voltammetry involves adding the sinusoidal waveform to the above voltage ramp and inputting to a potentiostat which applies the resulting waveform to the working electrode. The ac current response is fed into a lock-in amplifier where the in phase and out of phase components with the applied ac waveform of the current are separated and recorded on a data acquisition system. The out of phase current is plotted against the ramp voltage.
The working electrode may for example be circular and/or have a maximum surface dimension in any direction of 2pm to 1 mm, although dimensions outside this range are not precluded. Alternatively, the working electrode may be a microelectrode and preferably sized such that the mercury has a maximum surface dimensions of 2pm to 10pm in any direction. The or each working electrode may, for example, be circular with a diameter of 2pm to 10pm. Such dimensions serve to maximise the edge-to area and thereby enhance stability of the phospholipid layer.
The mercury layer may be formed by eletrodeposition. The amount of mercury deposited should be sufficient to give a continuous film of the mercury on the conductive carrier substrate. Increasing the thickness of the mercury layer will enhance the stability of that layer and allow repeated the mercury layer to allow repeated cycles of phospholipid layer deposition, sample measurement and removal of the phospholipid layer so that one electrode may be used many times without disruption of the mercury. Additionally thickness of mercury layer will be a consideration for use of the electrode assembly in a flow cell where the mercury layer is required to withstand forces associated with liquid flows through the cell. The amount of charge required for the electrodeposition process will depend on the required thickness of the mercury layer and this will in turn depend on factors such as the surface area of the conductive substrate (on to which the mercury is to be electrodeposited) and the concentration of mercury ions in the mercury deposition electrolyte.
Controlled electrodeposition of mercury from a solution containing Hg2+ ions (e.g. provided by Hg(N03)2) may be effected within an electrochemical cell. The mercury deposition electrolyte may contain HCIO4 which prevents oxidation of the Hg species. However the lipid interrogation will not take place in this acidic solution and will instead be in a more benign electrolyte such as 0.1 M KCI. Alternatively electrodeposition of mercury can be effected by pipetting a drop of the base electrolyte (e.g. HCIO4 + Hg(ll)) onto the surface of the first insulator. The electrodeposition may be facilitated by applying a potential of —0.4V vs. 3.5 M KCI Ag/AgCI to the working electrode surface in the deposition solution and recording the charge passed which is directly proportional to the mass of mercury deposited the deposition can be stopped by breaking the circuit. This allows precise control over the amount of Hg deposited.
Once the mercury layer has been deposited, the assembly can be transferred to a neutral electrolyte such as 0.1 M KCI and the electrode surface eiectrochemicaliy cleaned by applying an extreme negative potential <-2V evolving hydrogen gas to 'scrub' the surface free from organics.
The biosensor of the invention may be used, for example, for determining (i) the presence or otherwise in a sample of a species known to have biomembrane activity or (ii) whether or not a particular substance has biomembrane activity. The measurements may be made by voltammetry, (e.g. rapid cyclic voltammetry or ac- voltammetry effected in conventional manner such as along the lines disclosed in GB- A-2 193 326 (see for example Fig 1 thereof). RCV will be used for rapid interrogation of the sensor surface and in particular to determine the effect of the interaction of the two capacitance of the phospolipid which are particular sensitive to interactions. The invention has applications in a wide number of fields. These include:
(a) routine testing of drinking water for the presence of biomembrane active compounds which may be toxic agents;
(b) testing of potential pharmaceutical products for their biomembrane activity;
(c) detection of toxic gases and explosive vapours which (provided they interact with the monolayer surface) may be diagnosed using a multivariate analysis approach depending on the strength of the interaction with monolayers of varying chemical functionality; and
(d) environmental applications such as the in situ analysis of natural and marine waters for biomembrane active compounds such as (i) toxic biomembrane active peptides produced by blooms of cyanobacteria and dinoflagelates or (ii) pollutants.
It will be appreciated that the system may be calibrated for sensor application with a series of compounds eg polycyclic aromatic hydrocarbons, phenothiazine drugs and anti-microbial peptides which are known to modify the structure of phospholipid layers on electrodes. In each case the disruption/modification of the phospholipid layer has been well characterised electrochemically.
The present invention is particularly useful for analysing samples containing nanoparticles but may be used with other types of samples, e.g. as fully disclosed in the WO specification.
The invention is illustrated by way of example only with reference to the accompanying drawings, in which:
Figs 1 (a)-(i) illustrate one embodiment of biosensor apparatus in accordance with the invention in successive stages of operation; and
Figs 2 (a) and (b) show the results of the Example detailed below. The illustrated apparatus comprises a biosensor unit 1 (in the form of a flow cell) incorporating a bionsensor 2 of the type described more fully in WO 2009/016366 and comprising mercury immobilised on a conductive substrate platinum although other conductive substrate as disclosed in WO 2009/016366 may be employed. Biosensor unit 1 has a liquid inlet 3 and liquid outlet 4 associated with a single channel peristalitic pump 5 (available from Cole Palmer Instrument) located upstream of inlet 3 and control valves 6 and 7 which regulate supply and discharge of liquid to and from biosensor unit 1 along various flow paths in the manner described more fully below. Valves 6 and 7 are universal valve switching modules with six channels.
Further features of the illustrated apparatus are a syringe 8 located immediately upstream of the inlet 3 of the flow cell 1 and arranged to discharge its contents into the flow path leading to the inlet 3. Syringe 8 contains a phospholipid dispersion, the phospholipds being for the purpose of forming a monolayer on the mercury surface of the electrode in the biosensor 2 in the manner described more fully in WO 2009/016366.
A further syringe 9 is provided holding a sample to be analysed and is arranged to discharge a known (metered) amount of this sample into a reservoir 10 containing a known volume of buffered electrolyte.
Control electrolyte (buffered) is provided in a further reservoir 11.
Argon is fed to reservoirs 10 and 1 1 as depicted by the labelled arrows.
As indicated, biosensor 2 is of the general type described in WO 2009/016366 and, in the present case, comprises the working electrode (i.e. mercury deposited on conductive substrate) and a counter electrode. A separate Ag/AgCI reference electrode 12 is provided for the biosensor unit 1 , all three electrodes (working, counter and reference) being connected to a potentiostat 13 controlled by AUTOLAB software and associated with a monitor 14 for displaying results. Bionsensor unit 1 may be of a similar device to the flow cell illustrated in Fig 10 of WO 2009/016366 save that inlet port 406 of that flow cell is not required. Fig 1 (a) shows the apparatus in its "non-operational" condition in which there is no liquid circulation through the biosensor unit 1. In this "non-operational" condition reservoirs 10 and 1 1 are both charged with electrolyte, syringe 8 is charged with phospholipid dispersion and syringe 9 is charged with sample to be analysed.
In the first stage of operation (Fig 1 (b)), pump 5 is operated and valves 6 and 7 are set so that control electrolyte from reservoir 1 1 passes in sequence through valve 7, pump 5, biosensor unit 1 , valve 7, valve 6 and then to discharge, all as represented by the arrows shown in Fig 1 (b). At this stage, a trace may be displayed on monitor 14 showing the signal obtained from the bare mercury electrode (see Fig 1 (c)).
The plunger of syringe 8 may now be depressed to inject phospholipid dispersion into the flow line between pump 5 and inlet 3 of the biosensor unit 1 (Fig 1 (d)). Using potentiostat conditions described more fully in WO 2009/016366, a monolayer of phospholipid may be deposited on the mercury surface of the working electrode of biosensor 2. These conditions may involve using rapid cyclic voltammetry (RCV) employing an "up-and-down" voltage ramp between -0.2 and -3 V. Successful phospholipid monolayer formation on the mercury electrode may be checked using rapid RCV between -0.2 and -1.8 V and checking the standard current-potential profile (see screen trace depicted on monitor 14 in Fig 1 (d)).
In the next stage of operation (Fig 1 (e)), valves 6 and 7 are set so that electrolyte from reservoir 10 runs round a continuous flow path whereby this electrolyte from reservoir 10 is passed in sequence through valve 7, pump 5, bionsensor unit 1 , back to valve 7, to valve 6 and then returned to reservoir 10 (see arrows in Fig 1 (e)). Once the system has equilibrated, the plunger of syringe 9 is depressed (Fig 1 (f)) to discharge a known volume of sample into reservoir 10, the sample then circulating around the continuous flow path as described for Fig 1 (e).
The effect of the sample on the phospholipid monolayer deposited on the mercury working electrode may be viewed on monitor 14. A comparison of Figs 1 (e) and (f) has changed as a result of sample addition (see also fuller discussion of effects of sample of phospholipid layer given in WO 2009/016366). Once the measurement has been made, valve 6 is set (Fig 1(g)) so that the electrolyte (containing sample) previously circulating back to reservoir 10 is now discharged to waste as depicted by the arrows in Fig 1(g). Once electrolyte (containing sample) has been discharged, valves 6 and 7 are set (Fig 1 (h)) so that electrolyte from reservoir 11 passes through the apparatus as depicted by the arrows and is then discharged. During this stage, the mercury electrode may be "cleaned" by removal of the phospholipid using techniques as disclosed in WO 2009/016366. This may involve scanning the working electrode in a cathodic direction, e.g. over the range -0.2 V to -2.625 V @ 97 Vs"1 with the electrode being immersed in electrolyte so as to desorb any contaminating organic material into the bulk solution.
Finally, valve 6 is set (Fig 1 (i)) in readiness for a further cycle of the apparatus.
A significant advantage of the illustrated apparatus is that the separation of the sample (contained in syringe 8) from the phospholipid dispersion (contained in syringe 9) facilitates the injection of sample in electrolyte as sample with defined concentration and pH from buffer.
The illustrated apparatus is suitable particularly, but not exclusively, for measuring the effect of nanoparticle dispersions on the phospholipid layer.
The invention is further illustrated by the following non-limiting Example.
Example
Apparatus as illustrated in Fig 1 was used to determine the effect of LUDOX® Si02 nanoparticles of different particle sizes on a phospholipid layer deposited on the mercury electrode.
Two LUDOX® samples were tested, namely SM30 (with an Si02 concentration of 2.92 m ) and TM50 (with a SI02 concentration of 3.3 mM). The carrier electrolyte in reservoir 1 1 was 0.1 mol dm"3 KCI buffered with borate 0.1 mol dm'3/phosphate 0.25 mol dm"3 /tris 0.1 mol dm"3 buffers at pH 10.7. The same electrolyte was provided for reservoir 10. All samples were purified and redispersed in buffer prior to the tests.
Figures 2(a) and (b) display the results for nanoparticle biomembrane activity as a function of time in respect of the SM30 (0 and 4 min) and TM50 (0 and 6 min) respectively. The x axis label in Fig 2 is l/microamps. The nanoparticle activity is displayed as a depression of the two current peaks on the displayed RCV plot on the monitor. The activity is shown to depend closely on the nanoparticle dispersion tested.

Claims

1. A biosensor apparatus for analysing a sample, the apparatus comprising:
(vi) a biosensor unit having a liquid flow path therethrough between an inlet and an outlet of the unit, said unit comprising
(a) an electrode assembly comprising at least one working electrode comprised of a conductive carrier substrate having a surface coated with mercury immobilised on the surface of the substrate,
(b) a counter electrode, and
(c) a reference electrode,
(vii) a liquid supply arrangement for supplying a flow of electrolyte to the inlet of the biosensor unit,
(viii) a phospholipid supply arrangement for introducing phospholipid into electrolyte flow to the inlet of the biosensor unit for deposition on the exposed surface of the mercury,
(ix) a sample supply arrangement for introducing the sample to be analysed into electrolyte flow to the inlet of the biosensor unit, and
(x) means for determining variations in the differential capacitance of phospholipid deposited on the mercury as a function of potential against the counter electrode.
2. Apparatus as claimed in claim 1 wherein the sample supply arrangement is adapted to introduce a metered amount of said sample into the electrolyte flow.
3. Apparatus as claimed in claim 1 or 2 wherein, in the direction of electrolyte flow to the inlet of the biosensor unit, the phospholipid supply arrangement is downstream of the sample supply arrangement.
4. Apparatus as claimed in any one of claims 1 to 3 wherein the liquid supply arrangement is adapted selectively to supply either control electrolyte or electrolyte containing sample to the inlet of the biosensor unit.
5. Apparatus as claimed in any one of claims 1 to 4 wherein the liquid supply arrangement is adapted to provide a first flow path for supplying control electrolyte to the inlet of the biosensor unit and a second flow path for supplying electrolyte containing sample to the inlet of the biosensor unit.
6. Apparatus as claimed in claim 5 wherein the first flow path includes a first reservoir for holding control electrolyte.
7. Apparatus as claimed in claim 5 or 6 wherein the second flow path is adapted to operate as a continuous loop.
8. Apparatus as claimed in claim 7 wherein the second flow path incorporates a second reservoir for holding electrolyte and said arrangement for introducing the sample to be analysed into electric flow to the inlet of the biosensor unit introduces the sample into said second reservoir.
9. A method of analysing a sample to determine biomembrane activity (or otherwise) of the sample using the apparatus of any one of claims 1 to 8, the method comprising the steps of:
(iv) supplying the inlet of the apparatus a flow of electrolyte containing a phospholipid and effecting deposition of the phospholipid on the surface of the mercury remote from the carrier substrate, (v) supplying to the inlet of the apparatus a flow of electrolyte containing the sample to be analysed, and
(vi) using a voltammetric technique to determine the biomembrane activity.
10. A method as claimed in claim 9 wherein step (i) is effected by supplying control electrolyte to the inlet of the biosensor unit and adding phospholipid to the control inlet upstream thereof.
11. A method as claimed in claim 9 or 10 wherein in step (ii) the flow of electrolyte containing the sample to be analysed circulates continuously through the biosensor unit.
12. A method as claimed in any one of claims 8 to 11 wherein the sample to be analysed comprises nanoparticles.
EP11719034A 2010-03-09 2011-03-09 Biosensor apparatus and use thereof Withdrawn EP2545366A1 (en)

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