GB2231656A - Biosensor probe detecting chemical changes in liquids - Google Patents

Abstract

A biosensor probe for detecting certain chemical changes in liquids. The probe comprises an electrode 7 housed within a holder 3. One surface of electrode 7 is exposed 10a liquid sample in passageway 12 via a filter membrane 9. The membrane 9 is retained by means of a cup 4 having an aperture 10. In use, a sensitive layer is applied to that surface of the membrane facing the electrode 7 and electrical activity resulting from the reaction or otherwise of the liquid with the sensitive material is monitored by means of an electrical connection 8 to the electrode 7, and a further electrode 14. <IMAGE>

Description

"IMPROVEMENTS RELATING TO BIOSENSORS" This invention relates to biosensors and in particular to a biosensor probe for detecting certain chemical changes in liquids. The invention finds particular application in the detection of water pollution and in measuring changes in the composition of industrial process liquids - for example for monitoring fermentation vats.

The probe utilises a method of detecting chemical changes in liquids, developed by D.M. Rawson, and described in European Patent Application No.

0242225. The technique described in this patent application relies on immobilising or trapping bacteria, or other living cells, on a porous membrane which is immersed in the liquid being monitored.

Changes in the chemical composition of the liquid affect the activity of the living cells and this activity is monitored by measuring the electrical activity of the cells, preferably after stimulating the cells, for example by means of light. It will be seen that, with the correct choice of bacteria or cell type, a wide range of chemical changes can be monitored.

European Patent Application No. 0242225 describes in some detail a flow cell for monitoring pollutants in drinking water. Cells of a chosen bacteria are captured on a bacteriological filter and the filter, including cells, is held against a carbon electrode by means of a fine nylon mesh. A stimulator in the form of an LED is situated close to the cells.

Liquid to be monitored is caused to flow over the cells, and a further electrode is placed in the liquid flow downstream of the carbon electrode te complete the electrical circuit. Stimulation of the living cells on the filter membrane causes an electrical current to flow between the electrodes, and this current flow can be detected and measured. If the liquid is unpolluted, the cells continue living, and continue to exhibit substantially constant electrical activity; if, however, the liquid becomes polluted, the metabolic activity of the cells will alter, and it is even possible that some or all of the cells will die. In these circumstances the electrical activity of the cells will change or, in the event of cell death, may cease altogether. External electronics can detect such changes and alert personnel in the event of pollution.

The present invention seeks to provide an improved probe utilising the above-described techniques and which will have application not only in connection with water pollution, but in a wider field of biochemical sensing.

According to the invention the biosensor probe comprises an electrode assembly incorporating an electrode, cap means having an aperture therein, attachment means securely attaching said cap means to said electrode assembly in such a way as to leave a surface of said electrode exposed through said aperture and a filter membrane positioned to cover said exposed surface, the perimeter edge or edges of said membrane being trapped between the cap means and the electrode to thereby locate the membrane against the surface. In practice, some form of sensitive layer, for example living bacteria cells, will be applied to that surface of the filter membrane which faces the electrode. Thus, when the cap means is in position, the filter membrane becomes trapped against the electrode, with the sensitive layer sandwiched between the electrode and the filter membrane.It is of importance, in this arrangement, that the sensitive layer is in intimate contact with the electrode. In an alternative arrangement, the sensitive layer is formed as a sandwich between two separate filter membranes.

This has been found to be a useful way of confining the sensitive material, and also assists in the formation of a monolayer of cells (see later) which gives improved sensitivity; for the purposes of the present specification, however, it is assumed that the sensitive layer is sandwiched between the electrode and the filter membrane.

In use, the probe forms part of a complete biosensor which further includes a fluid flow pathway for a fluid to be monitored, means for securely locating the probe within said pathway so that the fluid can reach the sensitive layer, a further electrode to complete the electrical flow path with the first-mentioned electrode and, optionally, a light source for stimulating the sensitive layer to produce electrical activity - not all types of sensitive layer need such stimulation and, of those that do need stimulation, not all have to be stimulated by light.

Preferably the cap means is removably attached with respect to the electrode assembly so that the probe can be dismantled to insert a new filter membrane. Generally speaking, the sensitive layer will be applied to the membrane prior to fitting in the probe and, when the sensitive layer is finished with, perhaps because its activity is finished or it has become contaminated, the whole filter membrane including the sensitive layer, is disposed of, and a new filter membrane and sensitive layer fitted.

Another important feature is therefore ease of dismantling and ease of assembly, with particular reference to the requirement, mentioned above, that the sensitive layer be in intimate contact with the electrode assembly.

In one embodiment, the cap means is made of resilient material and has means whereby it may close over and grip the electrode assembly. Preferably the cap means is shaped and of a size which securely fits over the electrode assembly to removably attach the two together. Specific attachment means may also be provided: gripping means, for example ribs, positioned to stretch the material of the cap means as it is fitted, and thus ensure secure positioning.

Preferably the cap means is provided with a rebate around that edge of its aperture which faces the electrode, which rebate locates the membrane and is of sufficient depth to house the thickness of the membrane. In order to take into account tolerances in the formation of this rebate and, more particularly in the manufacture of the membrane, it is preferred that the depth of the rebate be less than the expected minimum width of the membrane by an amount sufficient to ensure that, for all dimensions within tolerance, the membrane protrudes slightly from the rebate. This ensures that, when the cap means is fitted, the membrane will be under slight compression around its trapped edge or edges so as to ensure the aforesaid intimate contact between the sensitive layer and the electrode. Typically, the membrane will be about 60 microns thick to a tolerance of + 10 microns. Taking also into account the tolerance on the rebate itself, a suitable rebate depth would be 45 microns.

Preferably sealing means are incorporated between the interior of the cap means and the electrode. This is to prevent the sensitive layer being flushed away due to flow of liquid through the filter membrane and into the joint between the interior of the cap means and the electrode. One method of providing a suitable sealing means, which is suitable where the electrode is made of a particularly soft material such as exfoliated graphite, is to form the interior surface of the cap means with an annular rib encircling the aperture and extending towards the electrode so that, when the cap means is fitted onto the electrode assembly, the rib digs into the surface of the electrode, thus effecting a secure seal.

In a preferred embodiment, the electrode assembly comprises a cylindrical holder, preferably of electrical insulating material, in which the electrode is fitted, with said surface exposed - means within the holder are provided for effecting electrical connection between the electrode and exterior circuitry.

In order that the invention may be better understood, an embodiment thereof will now be described by way of example only and with reference to the accompanying drawings in which: Figure 1 is a diagrammatic section through a biosensor incorporating the probe of the present invention; Figure 2 is a view similar to Figure 1, but showing a more practicable embodiment in more detail; and Figure 3 is an enlarged view of part of the apparatus of Figure 2.

Referring to Figure 1 there is shown a diagrammatic view of the biosensor intended to illustrate the principles of construction. The probe of the i-nvention is housed within a circular bore of a housing 2 and consists of an electrode holder 3 in the form of a piston moveable up and down within the bore 1 and a cap 4 of plastics material which fits over the narrowed end 5 of the holder with a friction fit. The holder has an axial bore 6 in which is held and retained at end 5 an electrode 7. The holder 3 is made from electrically insulating material such as polycarbonate. The electrode may, for example be made of exfoliated graphite. A connecting wire 8 is electrically joined to the electrode 7 and extends down the bore 6 to exterior electronics (not shown). The electrode 7 is positioned within its holder in such a way that one surface of the electrode is exposed.

Sandwiched between that surface and the inside of the cap 4 is a filter membrane 9 of, for example 0.2 micron pore size. In use of the apparatus, a sensitive layer is applied to that surface of the membrane 9 which faces the electrode 7. An aperture 10 in the cap 4 causes the opposite surface of the membrane 9 to be exposed to the exterior. The electrode holder 3 is sealed within the bore 1 by an 0-ring seal 11.

The bore 1 forms the leg of a T-shaped set of interconnecting passageways within the housing. The main fluid flow passage is indicated under reference 12, and the arrow A indicates the direction of flow.

Situated within the main passageway 12 is a light source in the form of an LED 13 and a further electrode 14 which with electrode 7 completes the electrical circuit.

Liquid to be tested is passed along passageway 12. The liquid passes through the filter membrane 9 and may or may not react with the sensitive layer. The electrical activity of the sensitive layer is monitored by measuring the potential difference and/or the current between the electrodes 7 and 14 and the external circuitry analyses the result. Taking a specific example of water pollution from the aforementioned European Patent Application 0242225, the sensitive layer could be composed of living cells, for example bacteria. Advantageously, the cells are applied as a mono layer. The cells are illuminated, possibly intermittently, by means of light from the LED 13. For this purpose, it is assumed that the filter membrane is transparent, or at least, translucent.The light stimulates the bacteria into activity, and the level of this activity is measured by detecting electron transfer from the cells to the electrode.

Electron transfer can be enhanced by using an electron transfer mediator added to the liquid to be tested.

The mediator may be potassium ferricyanide, dimethylbenzoquinone or p-benzoquinone either alone or in admixture; the preferred bacteria is cyanobacterium, for example synechococcus, or a Eubacterium, for example, E-coli.

The preferred types of filter membrane are the asymmetric or the symmetric anodic aluminium oxide membrane such as described in our European Patent Application 0178831. These are available commercially in various pore sizes, such as 0.2 micron, 0.1 micron and 0.02 micron. Such membranes comprise a plurality of parallel pores which extend from one face of the membrane to the opposite face, and open into both faces. The pores themselves may be parallel sided, or may have sides which are stepped so that the opening onto one surface is smaller than that into the opposite surface. Both types could be used for the present invention, depending upon the circumstances. The pore size can be controlled during manufacture, and is typically 0.2 microns for the present purpose.

Referring now to Figures 2 and 3 the probe will be described in more detail. Figure 2 is approximately equivalent to Figure 1, but it will be noted that the light source 13 and electrode 14 are not shown for clarity, although it is assumed that they will be present.

The more detailed view of Figure 2 shows a number of additional features not shown in Figure 1; The manner of electrically connecting the lead 8 is shown. The lead 8 (not shown in Figure 2) is inserted and soldered within a bore 15 of a connecting piece 16 made, for example, of brass. The outer surface of the connecting piece 16 is formed with sawtooth-shaped ribs 17 which dig into and grip the interior bore 6 of the electrode holder 3. The sawtooth shape of ribs 17 enables the connecting piece 16 to be pushed into bore 6 relatively easily, but reverse movement can only be achieved by damaging the holder.

Good electrical and mechanical contact with the electrode 7 is ensured by providing an upstand annular rib 18 on the connecting piece which digs into and securely grips the soft material of the electrode. A good area of electrical contact is desirable at that face of electrode 7 adjacent connecting piece 16 since the preferred material of the electrode - exfoliated graphite - is strongly anisotropic, exhibiting much higher resistance to the flow of current in the vertical direction in Figure 2, than in the horizontal direction.

A further feature not illustrated in Figure 1 is that the bore 1 is stepped to define a shoulder 19, against which the probe can be pressed by means of a nut (not shown) threaded onto that part 20 of the bore 1 behind the probe, which is threaded. This ensures that the probe is always accurately and firmly located in housing 1, and also ensures that the cap 4 is fully pushed home over the end of the electrode housing 3.

It will be noted that the housing 3 is also stepped, to provide a shoulder 21 which defines, with the lower edge of the skirt of cap 4, a circumferential notch in which the 0being 11 is seated.

The construction of the cap 4 will now be described in more detail with reference to Figures 2 and 3. It is to be noted that Figure 3, although showing part of Figure 2 at a greater magnification, is not in itself drawn to scale, as will be apparent when it is realised that a typical width of filter membrane 9 is 60 microns.

The cap 4 is cup shaped and comprises a planar portion 22, having the aperture 10 therein, and a skirt portion 23 depending therefrom. The cap 4 is made from resilient material, such as polypropylene, so that, as the cap is pushed over the end 5 of the electrode housing 3 the skirt portion 23 stretches slightly so that tight gripping of the cap 4 on the housing is ensured. A circumferential bead 24 is formed on the exterior surface of the housing 3 to accentuate this stretching, and thus improve the grip.

The inner surface of the planar portion 22 is formed with an upstanding ridge 25, typically about 0.3mm high, which is of annular shape and concentric with the aperture 10. The purpose of this ridge is to dig in to the soft material of the electrode 7 as the cap is pushed home to provide a seal around the edge of the aperture to prevent the sensitive layer from being washed away. The inner surface of the planar portion 22 is also rebated around the edge of the aperture 10 in order to provide a locating seating for the filter membrane 9. This rebate is shown in Figure 3. The depth of the rebate (i.e. its extent in the axial direction of the probe) is dictated by the total thickness of the membrane 10 and any sensitive layer 26 applied thereto. Often the sensitive layer is of such thickness, for example 0.5 microns, that it can be ignored.The tolerance in the thickness both of the membrane and in the depth of the rebate also have to be taken into account. The object is to size the depth of the rebate such that, when the membrane 9 is seated in it, the membrane will always stand slightly proud of the inner surface of the planar portion 22 of the cap 4. This ensures that, when the cap 4 is pushed home, the edges of the membrane 9 within the rebate will be slightly compressed to ensure an intimate contact between the sensitive layer 26 and the electrode 7.

Typically, for a membrane of thickness 60 microns + 10 microns, the depth of the rebate will be set at 45 microns.

The probe is positioned within bore 1 so that it is set back slightly from the full flow along passageway 12. Typically the distance B is about 3mm., the diameter of the passageway 12 being, for comparison, about 5mm..

In use, liquid to be tested is caused to flow along passageway 12. Liquid flows up the bore 1 and through the pores of the membrane 9 from one face to the other. When the liquid reaches that face of the membrane which faces the electrode 4, the liquid comes into contact with the sensitive layer 26 and reacts therewith, or not, in the manner described above. The electrical activity within the sensitive layer is monitored by measuring the current flow between electrodes 4 and 14 using external circuitry.

Leakage of liquid beyond the sensitive layer into the interface between the inside surface of the planar portion 22 of the cap 4 and the electrode 7 is prevented by the ridge 25. It may be necesary to provide a small slot across the ridge 25, and possibly across the ridge 24 as well, at one point in order to permit passage of air during fitting of the cap 4 over the housing 3. Otherwise there may be a pressure build up of air in the closing stages of pushing the cap 4 home which might prevent the user from fully pushing the cap 4 into place.It is believed that such slots should be capable of being designed in such a way as to allow escape of trapped air during assembly without affecting the sealing performance once assembled; however, even if the seal is, as a result, not complete, it is considerably better than no seal at all, and will effectively prevent the sensitive layer being flushed out in the manner described above.

In practice, the liquid flow along passageway 12 is monitored continuously or intermittently over a period of time, and therefore the probe will be in place for quite some time, and its electrical output is monitored either continuously or intermittently during this time for any changes in the composition of the liquid. There is no or only a very small flow of liquid through the bore 1 or through the pores of membrane 9, and changes in the composition of the liquid reach the sensitive layer by diffusion from the passageway 12.

The probe may stay in place for as long as the sensitive layer remains sensitive. Once the sensitivity is gone, either due to ageing or contamination, the membrane 9 must be replaced for a new one having a fresh sensitive layer immobilised thereon. During the changeover period, liquid being tested can be diverted through an alternative biosensor so that continuous monitoring is maintained. The present invention does however provide a quick and easy way of fitting the sensitive layer so that the biosensor can be assembled very quickly. Likewise, during use, the sensitive layer can be readily changed for a new one so that the biosensor can be refreshed very quickly.

Claims (14)

1. A biosensor probe comprising an electrode assembly incorporating an electrode, cap means having an aperture therein, attachment means securely attaching said cap means to said electrode assembly in such a way as to leave a surface of said electrode exposed through said aperture and a filter membrane positioned to cover said exposed surface, the perimeter edge or edges of said membrane being trapped between the cap means and the electrode to thereby locate the membrane against the surface.

2. A biosensor probe as claimed in claim 1 further comprising a sensitive layer applied to a surface of said filter membrane.

3. A biosensor probe as claimed in claim 2 wherein said sensitive layer is applied to that surface of said filter membrane which faces the electrode so that the sensitive layer is sandwiched between the electrode and the filter membrane.

4. A biosensor probe as claimed in claim 2 including a further fitter membrane, and wherein said sensitive layer is positioned between said filter membrane and said further filter membrane.

5. A biosensor probe as claimed in any one of the preceding claims wherein the cap means is removably attached with respect to the electrode assembly.

6. A biosensor probe as claimed in any one of the preceding claims wherein the cap means comprises a wall in which is situated said aperture and from which depends a skirt portion of resilient material operable to grip the electrode assembly to retain the cap means in place.

7. A biosensor probe as claimed in claim 6 wherein the inside surface of said skirt portion and/or the co operating outer surface of said electrode assembly includes gripping means to assist retention of said cap means on the electrode assembly.

8. A biosensor probe as claimed in either one of claims 6 or 7 wherein that surface of the wall facing the electrode is provided with a rebate opening into the aperture for locating the filter membrane or membranes.

9. A biosensor probe as claimed in claim 8 wherein the depth of said rebate is less than the expected thickness of the filter membrane or membranes so that, when the cap means is fitted, the edges of the membrane or membranes within the rebate are subjected to a slight compressive stress.

10. A biosensor probe as claimed in any one of claims 6 to 9 further comprising sealing means between the interior of the cap means and the electrode.

11. A biosensor probe as claimed in claim 9 wherein said sealing means comprises a rib projecting from the inside surface of said wall and substantially surrounding said aperture, said rib being operable to dig into the material of said electrode when the cap means is closed over the electrode assembly.

12. A biosensor probe as claimed in claim 11 wherein said rib is provided with a transverse passage or passages sufficient to permit venting of air, but not sufficient to permit significant leakage of liquid.

13. A biosensor comprising a fluid flow pathway for fluid to be monitored, a biosensor probe as claimed in any one of the preceding claims and having applied to a surface of said filter membrane a layer of material sensitive to the fluid to be monitored, means for securely locating the probe within said pathway so that fluid can reach the sensitive layer, and a further electrode to complete the electrical flow path with the first-mentioned electrode, the arrangement being such that a parameter of the sensitive layer, which may be altered by interaction with the fluid to be monitored, is such as to alter the electrical characteristics between the two electrodes.

14. A biosensor as claimed in claim 13 further comprising a light source for stimulating the sensitive layer to produce electrical activity.