GB2161274A - Method and apparatus for measuring enzyme concentrations - Google Patents

Abstract

The concentration of enzymes, bacteria or other related populations in a first solution 12 is determined by imposing a potential between a first electrode 56 in the first solution and a second electrode 52 in a second solution 13 in electrolytic connection 100 with the first solution & measuring the current flow; the first electrode being of graphite of porosity such that absorption on its surface penetrates to less than 6mm (<10% porosity). The electrode is preferably cylindrical, coated on its sides with impermeable insulating material with its end exposed and is rotated at 100-2000 RPM. The first solution further contains a redox reagent, an enzyme substrate & optionally disodium EDTA and the enzyme oxidises or reduces the redox reagent which is restored to its original oxidation state by the measured current. <IMAGE>

Description

SPECIFICATION Method and apparatus for measuring enzyme concentrations This invention relates to methods and apparatus for determining concentrations of active populations of enzymes or enzyme-containing populations, such as bacteria and yeasts.

Traditionally, such populations are counted by microscopic techniques or by more sophisticated techniques, such as the measurement of optical turbidity of liquids containing the populations. US A-3506544 described a technique for measuring the concentration of certain constituents of enzymecatalyzed reactions using an electrochemically-reversible redox couple. The enzyme, a redox couple such as methylene blue, a substrate such as glucose, and a compatible conductive medium, such as buffered water solution, were mixed in an electrolytic cell through which a current was passed by impressing a voltage across a pair of electrodes.

The electrodes were typically made of noble metal.

The activity of the enzyme reduced the oxidized redox couple at a rate corresponding to the concentration of the enzyme. The reduced redox couple was subject to reoxidization at the anode of the cell, which thus produced a cell current proportional to the concentration of enzyme in the solution.

The various prior art techniques for measuring enzymes or bacteria populations suffered certain deficiencies. Some were not sufficiently sensitive to detect very low concentrations, or were very slow, or required too many steps to make the determinations efficiently. Some were also quite ex pensive especially those which used noble metal or similar electrodes. Also, it was found that unex plainable errors sometimes occurred in the analysis of a particular specimen.

Another problem with prior art devices surfaced when the same apparatus was used consecutively for multiple specimens. It was found that moving the electrodes from one specimen cell to another, contaminated the second cell or specimen with enzymes, or other components that had been adsorbed by the electrodes. Also, great care was required to ensure that the cell was filled to very precisely measured depths, identical to those used to calibrate it, in order to ensure that electrode surface areas submerged in the electrolyte remained constant. Even with great care in this regard, however, unexplainable errors in measurements occu rred.

A further problem of the prior art was encountered due to the need to deaerate the solution prior to running the test. It was found that, under some circumstances, unpredictable amounts of aerobic bacteria might be deactivated, due to the deaeration step. On the other hand, if the solution was not deaerated, the oxygen remaining interfered with the precision of the measurement, since it contributed to the reaction at the anode upon which the measurement was based.

These and other problems of the prior art techniques can be greatly alleviated or eliminated in accordance with the present invention.

According to a first aspect of this invention a method of measuring the concentration of an enzymatic agent in a first solution comprises at least partially submerging a first electrode in the first solution, at least partially submerging a second electrode in a second solution which is in electrolytic communication with the first solution, and imposing an electric potential across the first and second electrodes, the resulting current flow between the electrodes being related to the concentration of the enzymatic agent in the first solution, the first electrode comprising a graphite conductor having a porosity sufficiently low to prevent significant absorption of the solution more than substantially 6 mm beneath its surface.

According to a second aspect of this invention a method of making an electrode for at least partially submerging in a solution and making precision measurements of current flow through it comprises providing a cylindrical graphite conductor having a diameter in a range from substantially 1 mm to substantially 100 mm and a porosity less than substantially ten percent, coating the cylindrical surface of the conductor with a polymeric material which is impermeable to the solution, and flattening, smoothing, and polishing the end surface of the conductor to a metallic lustre.

The methods in accordance with this invention permit high precision and reproducibility of measurements, and also make it possible to use the same electrode for a series of consecutive measurements of concentrations of enzymatic agents in different solutions simply by removing the exposed end portion of the electrode between successive measurements.

A particular example of a method in accordance with this invention will now be described with reference to the accompanying drawings; in which: Figure 1 is a partly sectioned elevation of apparatus for measuring enzymatic agent concentrations; Figure 2 is a graph of the current flow through the sensing circuit plotted against time; Figure 3 is a graph of the relationship between reaction rate and bacterial concentration during solution testing; and, Figure 4 is a cross section through the graphite electrode taken along lines 4-4 shown in Figure 1.

Figure 1 shows an actinic glass container 10 containing a solution 12 whose concentration of enzymatic agent is to be tested. The enzymatic agent may comprise an enzyme per se or a yeast; however, the utility of the invention is especially contemplated for solutions containing bacteria.

Solution 12 contains water which is buffered using suitable buffering agents, such as potassium dihyrogen phosphate (KH2PO4) and dipotassium hydrogen phosphate (K2HPO4) to a pH of 7.0. The optimum pH depends upon the particular enzymatic agent being tested, but typically, a neutral pH of 7.0 is satisfactory. Disodium salt of ethylenediaminetetraacetic acid (disodium edetate or disodium EDTA) is added in an amount sufficient to render its concentration about 0.01 molar in the solution 12.This has been found to reduce the interference of metal ions, which may be present in the solution as impurities, and which would otherwise tend to affect the enzymatic activity or the current flow through solution 12 and produce erroneous results. it is believed that the disodium EDTA forms complexes with the metal ions to prevent their precipitation at process conditions (which precipitation might otherwise occlude the bacteria or other enzymatic agent being analyzed).

Thus, the effective and optimum amounts of disodium EDTA will depend on the metal ions present and can be determined by routine experimentation.

A substrate and redox couple is added to the solution. The substrate and redox couple chosen should be of a type which is subject to catalytic action from the particular enzymatic agent being tested. Typically, for most bacteria, excellent results are achieved using a solution 12 which contains approximately 0.01 molar concentration of glucose and approximately 0.0001 molar concentration of methylene blue (methylthionine chloride).

Access to the interior of container 10 is obtained through necks 4, 16, 18, and 20. After the contents are all in place, an inert gas, such as argon, is injected through line 30 and dispensing tube 32, bubbling out through openings 34 and up through solution 12. This procedure is typically followed for about five minutes to purge most of the free oxygen from solution 12. The bubbling argon and any volatile materials, such as oxygen, pass upwardly through vapour space 35 and are removed from container 10 by means of outlet line 36. Both the inlet line 30 and the outlet line 36 are sealingly fitted through a two-hole stopper 38 within access neck 14.

A separate glass container 11 containing a solution 13 is electrolytically connected to the system in container 10 by means of salt bridge 100, consisting of agar and potassium chloride. The solution 13 contains an appropriate electrolyte, typically a 0.1 molar ferric EDTA solution, or alternatively, an aqueous potassium chloride solution.

Access to the interior of container 11 is obtained throug necks 15, 17 and 19. The solution 13 may also be deaerated, if desired, by injecting an inert gas, such as argon, through inlet line 31 and dispensing tube 33, bubbling out through opening 37 and up through the solution 13. The bubbling argon and oxygen, and any other volatile materials, pass upwardly through vapour space 39 and are removed from container 11 by means of outlet line 41. The inlet line 31 and outlet line 41 are both sealingly fitted through a two-hole stopper 43 within access neck 15.

A battery or DC power supply 40 is provided with a potentiometer 42, having a potentiometer slider 44 and resistance means 46. The slider 44 is connected by means of conductor 50 to counterelectrode 52. The counterelectrode 42 may be made of any convenient electrode material. For most usages, it is found that an iron electrode is satisfactory. The counterelectrode 52 is sealingly fitted within access neck 17 by means of stopper 54.

A high density, low porosity graphite electrode 56 is fitted within access neck 20 of container 10 by means of stopper 57 and is connected by conductor wire 58 through ammeter 60 and conductor 62 to the positive side of the battery 40.

A reference electrode 70, such as a saturated calomel electrode, is fitted through stopper 72 in access neck 18 of container 10 and connected by conductors 74 and 76 through volt meter 78 to the conductor 58 from the graphite electrode.

To place the apparatus in operation, it is important to rotate the graphite electrode 56 to ensure representative contacting of the electrode with the constituents of solution 12 to replace the redox couple being consumed at the surface of the electrode. This is achieved using a drive means 80 and gear mechanism 82, shown schematically in Figure 1. A range of speeds of rotation may be successfully employed; however, it is preferable to rotate the electrode at speeds of about 100 to 2,000 RPM.

Lower speeds can be used; however, at too low speeds, the electrode becomes more sensitive to external vibrations and less sensitive to detecting low concentrations of bacteria.

To start a test procedure, the potentiometer slider 44 is adjusted along resistance means 46 until voltmeter 78 is at essentially a 0.0 reading. After or simultaneously with the purging of the dissolved oxygen in solution 12 with argon, any remaining traces of oxygen are preferably removed by adding, for example, enough ferrous EDTA to reduce the measured current flow through ammeter 60 to essentially zero.

The current flow through the test circuit (i.e., between graphite electrode 56 and counterelectrode 52), as measured by ammeter 60, is plotted as shown in Figure 2. Typically, the change of current with time is initially nonlinear, but after a short period of time a linear relationship is established. The slope of the linear portion of the line (indicated by the tangent line in Figure 2) is proportional to the reaction rate of the redox couple at electrode 56, due to the catalytic action of the enzymatic agent.

A typical relationship between the reaction rate and the concentration of bacteria in a solution saturated with glucose and methylene blue (relative to the amount of bacteria available) is shown in Figure 3.

This example was performed using a 300 ml flood water sample in container 10 with 0.112 grams of disodium EDTA to make the solution 0.001 molar in disodium EDTA. To this solution, 1.633 grams of KH2PO4 and 3.136 grams of K2HPO4 were added to make the solution 0.1 molar in phosphate. The pH was adjusted to seven using 0.5 Normal NaOH and 0.5 grams of glucose (0.01M), and 15ml of 0.01M methylene blue (5 x 10"M) were added to the solution. The solution was then placed in contact with the electrodes; the rotating electrode having been previously cleaned by polishing with fine sandpaper and then polished on a glass plate. The carbon electrode had an exposed lower surface of 0.30 cm2 and was rotated at 1,000 RPM.

The voltage was set so that the rotating electrode was at 0.00 volts vs. the saturated calomel electrode. The current was measured as a function of time. When the slope of the change in current with time became constant, the slope was determined and the number of bacteria calculated, based upon the calibration curve. In this example, the reaction rates determined from the slope of the tangent line in Figure 2 were used, as shown by the broken lines in Figure 3, to establish that the bacteria concentration was between 106 and 107 cells per millilitre of solution when the reaction rate was approximately 6.25 x 10-9.

Graphs similar to Figure 3 can be prepared using standard concentrations of bacteria or other enzymatic agents at standard test conditions. After this calibration curve has been prepared, it can be used repeatedly for tests on different samples of solutions containing the same or similar bacteria or enzymatic agents.

Figure 4 shows a cross section taken along lines 4-4 of the high density, low porosity graphite electrode 56 shown in Figure 1. The electrode 56 consists of a graphite core 96 which typically consists of a very high purity, impermeable cylindrical element ranging from about 1mm to about 100 mm, preferably from about 3 mm to about 10 mm, in diameter. The graphite should have a porosity less than about 10%. This is important to minimize absorption of solution which might then be carried over into a subsequent sample being tested. Also, any absorption into interstices of pores of an electrode has an electrical effect analogous to increasing the surface of the electrode, i.e., it increases the rate of oxidization of the redox couple and may produce erroneous readings, especially at low speeds of electrode rotation.

The coating 98 around the exterior surface of the cylindrical rod 96 can be of any convenient impermeable material. Preferably, a polymeric material (such as epoxy resin, polyurethane, or the like) is used, since the graphite rods can be dipped in such materials and the adhering coating can be cured in a relatively short time to form a tough, dense, protective layer. The layer need not be excessive in thickness, provided it is impervious to the liquid and acts as an insulator against substantial current flow relative to the conductivity of the graphite. Generally, epoxy coatings about 1 mm in thickness have proven acceptable.

After the coating 98 is applied, the end portion is removed to expose an uncoated graphite surface 110 (see figure 1) at the end of the electrode. Surface 110 thus constitutes the only area of contact between the graphite and the solution .2, the protective coating 98 running at least up to the vapour space 35 of the container 10 and preferably covering the entire graphite portion of the electrode.

The end surface 110 should be ground or sanded, e.g., with very fine sandpaper, to remove any scratches and provide a flat surface. It is preferably then polished using smooth hard paper on a hard flat surface, such as a glass plate, until a metallic lustre covers the entire surface 110. The electrode is then rinsed with methanol and distilled water to remove any oils or other contaminants just prior to use.

By providing a very smooth, glossy, impermea ble surface 110, high precision can be obtained in correlating consecutive measurements, since highly reproducible electrical circuits having pre cise exposed surface areas of the graphite electrode can be provided.

A particularly attractive feature of the coated graphite electrode of this invention is its utility in performing consecutive tests on different speci mens or samples of solution 12. For example, the solution can be changed or the electrode can be withdrawn from access neck 20 and used in a different container 10 with a different solution. Prior to inserting the graphite electrode 56 in a new so lution, the lower end portion is removed (for example, by grinding a portion away) to ensure that even minor quantities of absorbed solution from the previous test have been removed. It has been found that by grinding off about 6 mm or more of the bottom of the electrode, and then sanding and polishing the bottom surface, the electrode is suitable for use with results indistinguishable from a new electrode. Moreover, unlike prior art electrodes which used, e.g., noble metals, it has been found that any deposits or contamination of the outside coating 98 of the electrode do not affect the precision of the test. Thus, it is not necessary to discard the electrode when its outer surface becomes corroded or coated with extraneous agents.

Claims (19)

1. A method of measuring the concentration of an enzymatic agent in a first solution comprising at least partially submerging a first electrode in the first solution, at least partially submerging a second electrode in a second solution which is in electrolytic communication with the first solution, and imposing an electric potential across the first and second electrodes, the resulting current flow between the electrodes being related to the concentration of the enzymatic agent in the first solution, the first electrode comprising a graphite conductor having a porosity sufficiently low to prevent significant absorption of the solution more than substantially 6 mm beneath its surface.

2. A method according to claim 1, wherein the graphite conductor has a porosity less than substantially ten percent.

3. A method according to claim 1 or 2, wherein the first electrode is cylindrical and is rotated about its longitudinal axis at a speed sufficient to ensure that it makes representative contact with the constituents of the first solution.

4. A method according to claim 3, wherein the speed is in a range from substantially 100 RPM to substantially 2,000 RPM.

5. A method according to claim 3 or 4, wherein the cylindrical first electrode is between substantially lmm and 100 mm.

6. A method according to any one of the preceding claims, wherein the submerged portion of the first electrode is coated over its entire surface, except its submerged end surface, with an electrically insulating material which is impermeable to the first solution.

7. A method according to claim 6, wherein the first electrode is between substantially 3 and 10 mm in diameter.

8. A method according to any one of the preceding claims, wherein the first solution comprises an aqueous solution containing enzymatic agent, a redox couple and substrate, and which further comprises the step of adding a sufficient amount of disodium EDTA substantially to improve the precision of the measurement of concentration of the enzymatic agent.

9. A method of making an electrode for at least partially submerging in a solution and making precision measurements of current flow through it comprising: providing a cylindrical graphite conductor having a diameter in a range from substantially 1 mm to substantially 100 mm and a porosity less than substantially ten percent, coating the cylindrical surface of the conductor with a polymeric material which is impermeable to the solution, and flattening, smoothing, and polishing the end surface of the conductor to a metallic lustre.

10. An electrode prepared by the method of claim 9.

11. A method of measuring enzyme concentration in an aqueous solution containing the enzyme and dissolved oxygen comprising: placing a sample of the solution in a container, buffering the solution to a substantially neutral pH, adding a substrate and redox couple to the solution, the substrate and redox couple being subject to a shift from one oxidation state to another by catalytic action of the enzyme, removing the dissolved oxygen from the aqueous solution after the substrate and redox couple have been added to it, providing a separately contained electrolytic solution, electrolytically connecting the aqueous solution and the electrolytic solution, at least partially submerging a counterelectrode in the electrolytic solution, at least partially submerging a graphite electrode in the aqueous solution, the graphite electrode comprising a cylindrical graphite conductor having a diameter in a range from substantially 1 mm to substantially 100 mm and a porosity of less than substantially ten percent, the conductor having an insulating coating over its cylindrical surface, the coating being impermeable to the aqueous solution, the end surface of the cylindrical conductor being free of the coating and being flat, smooth, and polished to a metallic lustre, rotating the graphite electrode about its longitudinal axis at a speed in a range from substantially 100 to substantially 2,000 RPM and imposing an electric potential between the graphite electrode and the counterelectrode, measuring the current flow between the counterelectrode and the graphite electrode, and determining the enzyme concentration in the aqueous solution from the rate of change of the current flow.

12. A method according to claim 11, wherein the counterelectrode comprises iron.

13. A method according to claim 11 or 12, wherein the enzyme comprises bacteria and the substrate and redox couple comprise glucose and methylene blue.

14. A method according to claim 11, 12 or 13, wherein metal ions are present in the aqueous solution as impurities, and further comprising the step of adding sufficient disodium EDTA to the solution to minimize interference of the metal ions with the precision of measurements of the enzyme.

15. A method according to any one of claims 11 to 14, further comprising removing at least substantially 6 mm of the submerged end of the graphite electrode after measuring the current flow through it; flattening, smoothing, and polishing the resulting newly exposed end surface to a metallic lustre; and reusing the graphite electrode.

16. A method according to any one of claims 11 to 15, wherein the dissolved oxygen is removed from the aqueous solution by bubbling an oxygenfree inert gas through it.

17. A method of measuring the concentration of an enzymatic agent substantially as described with reference to the accompanying drawings.

18. A method of making an electrode substantially as described with reference to the accompanying drawings.

19. An electrode substantially as described with reference to the accompanying drawings.