FR2567268A1 - METHOD AND ELECTRODE FOR MEASURING ENZYMATIC CONCENTRATIONS AND METHOD FOR PREPARING SUCH AN ELECTRODE - Google Patents

Abstract

METHOD AND ELECTRODE FOR MEASURING ENZYMATIC CONCENTRATIONS AND PROCESS FOR PREPARING SUCH AN ELECTRODE; TO DETERMINE THE CONCENTRATIONS OF ENZYMES, BACTERIA AND OTHER RELATED POPULATIONS, THESE POPULATIONS ARE USED TO CATALYZE THE REDOX TORQUE REDUCTION, THEN THE REDUCED TORQUE IS OXIDIZED IN AN ELECTROLYTIC CELL 12 USING A DENSE GRAPHITE 56 ELECTRODE 96 PARTLY COATED WITH A LAYER 98 OF A WATERPROOF INSULATION MATERIAL; THE ELECTRICITY NECESSARY TO REOXIDIZE THE TORQUE IS RELATED TO THE CONCENTRATION OF THE ENZYMES OR OTHER ACTIVE POPULATIONS.

Description

l

  The present invention relates to a method and an

  trode for measuring enzyme concentrations and a method

  for preparing such an electrode. More particularly the

  vention relates to a method and an apparatus for determining the concentrations of active populations of enzymes or populations containing enzymes, such as bacteria, yeasts or the like. Conventionally, these populations are counted according to microscopic techniques or according to more sophisticated techniques, such as the measurement of the optical turbidity of liquids containing the populations. United States patent No. 3,506,544 describes a technique for measuring the concentration of certain constituents of enzymatic catalysis 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 a buffered aqueous solution, are mixed in an electrolytic cell through which a current is passed by application of a voltage between a pair of electrodes. The electrodes are typically made of a noble metal. The activity of the enzyme reduces the oxidized redox couple at a speed which corresponds to the concentration of the enzyme. The reduced redox couple is subjected to reoxidation at

  the cell anode, which produces a proportional cell current

  tional concentration of the reduced redox couple. The rate of increase of the current thus proves to be proportional to the

  concentration of the enzyme in the solution.

  The various techniques of the prior art, for measuring the populations of enzymes or bacteria, have certain shortcomings. Some are not sensitive enough to detect very low concentrations, or are very slow, or require too many stages to allow effective determinations. Some are also very expensive, especially

  when using electrodes of noble metals or the like.

  We also found that inexplicable errors appear

  sometimes in the analysis of a particular sample.

  Another problem appears with the

  prior art when the same apparatus is used consecutively

  cutive with- multiple samples. We can see that the displacement

  cementation of the electrodes, from one sample cell to another, contaminates the second cell or the second sample with

  enzymes- or other components that have been absorbed by the elect

  trodes. Also care must be taken to ensure that the cell is filled to a very precise level, identical to that used for its calibration, so that the surface of the electrode immersed in the electrolyte remains constant. However, even when great care is taken in this regard, inexplicable errors by

measurements happen.

  Another problem encountered in the prior art is

  the need to deaerate the solution before performing the analysis.

  It has been found that, in certain circumstances, unpredictable amounts of aerobic bacteria can be deactivated as a result of the deaeration stage. On the other hand, if the solution is not deaerated, the remaining oxygen harms the accuracy of the measurement, because it contributes to the reaction at the level of the anode on which rests

measurement.

  These prior art problems, as well as others, can be greatly reduced or eliminated according to the invention. The invention relates to a method and a device for measuring the concentration of an enzymatic agent in a solution.

  in which an electric current is passed between a counter -

  very dense, cylindrical, elongated, low porosity graphite electrode. The graphite electrode rotates around its longitudinal axis in the solution which also contains a

  redox couple and a substrate which are catalyzed by the enzymatic agent

  tick to react and modify the redox couple to pass it from one oxidation state to another. The variations in the oxidation state cause a change in current in the rotating electrode, this change being linked to the speed of variation of the oxidation state of the redox couple which itself is linked to the concentration of l enzyme agent. The submerged portion of the graphite electrode is coated over its entire surface, except for the end surface, with an insulating protective layer, such as a layer of epoxy resin which is non-conductive and is inert and impermeable towards the constituents of the solution. This allows very precise and reproducible measurements and also the use of the same electrode for a series of consecutive measurements of concentrations of enzymatic agents in different solutions, by simple elimination of a portion.

  from the apparent end of the electrode between successive measurements.

  The method also contemplates deaeration of the solution to remove oxygen before measuring the intensity of the current. For certain enzymatic agents, such as aerobic bacteria, it has been found that the precision is greatly increased by introducing the redox couple and the substrate into the solution before

the deaeration stage.

  In its preferred embodiments, the invention includes the use of disodium EDTA to significantly improve the

analysis accuracy.

  Other characteristics and advantages of the invention

  will be understood on reading the description which follows and teaches

  Referring to the accompanying drawings, in which: FIG. 1 illustrates an apparatus for measuring concentrations of enzymatic agents according to the invention; FIG. 2 graphically illustrates an example of the intensity of the current through the detection circuit as a function of time; FIG. 3 graphically illustrates the relationship between the reaction rate and the concentration of bacteria, during the analysis of a solution; and Figure 4 is a cross section of the graphite electrode, taken along lines 4-4 of Figure 1:

  The invention will now be described in detail.

  The figure illustrates a container 10 made of actinic glass containing a solution 12, the concentration of enzyme agent of which must be determined. The enzyme agent may consist of an enzyme or a yeast; however the invention is particularly

  useful in the case of solutions containing bacteria. -

2567268.

  Solution 12 contains Water which is buffered by the use of suitable buffering agents, such as potassium dihydrogen phosphate (K2HPO4) and dipotassium hydrogen phosphate (K2HPO4)

  at a pH of 7.0. The optimal pH depends on the specific enzyme agent

  culier to be analyzed, but typically a neutral pH of 7.0 is

  satisfactory. The disodium salt of ethylenediamine acid is added.

  tetraacetic (disodium edetate or disodium EDTA) in an amount sufficient for its concentration to be approximately 0.01 M in solution 12. This has been shown to reduce interference from metal ions which may be present in the solution as impurities and which would otherwise tend to modify the enzymatic activity or the intensity of the current in solution 12 and to produce erroneous results. It seems that disodium EDTA forms complexes with metal ions by avoiding their precipitation under the operating conditions (this precipitation could otherwise

  coat the bacteria or other enzyme agent to be analyzed).

  -So the effective and optimal amounts of disodium EDTA depend on the metal ions present and can be determined by

simple experiences.

  A substrate and a redox couple are added to the solution.

  The substrate and the redox couple chosen must belong to a type sensitive to the catatytic action of the particular enzymatic agent to be analyzed. Typically, for most bacteria, excellent results are obtained by using a solution 12 which contains a glucose concentration of approximately 0.01 M and a methylene blue concentration (methylthionine chloride) of approximately 0.0001 M. Access to the interior of container 10 is via the flasks 14 , 16, 18 and 20. When all the contents are in place, an inert gas is injected, such as argon through the pipe 30 and the distributor tube 32, so that it escapes through the openings 34 and s' pupil by dabbling in solution 12. This operation

  typically takes about 5 minutes to purge the major -

  part of the free oxygen in solution 12. The bubbling argon and any volatile matter such as oxygen, rise through

  the vapor space 35 and are eliminated from the container 10 by the canali-

  outlet 36. The inlet pipe 30 and the outlet pipe 36 are tightly secured by means of a plug

  with two holes 38 placed on the access neck 14.

  A separate glass container 11 containing a solution 13 is electrolytically connected to the container system 10, by means of a salt bridge 100, consisting of agar and potassium chloride. Solution 13 contains a suitable electrolyte, typically a 0.1 M solution of ferric EDTA, or otherwise a solution

aqueous potassium chloride.

  Access to the interior of the container 11 is via the necks 15, 17 and 19. The solution 13 can also be deaerated, if desired, by injection of an inert gas such as argon, by the inlet pipe 31 and the distributor tube 33, so that it leaves through the openings 37 and bubbles up into the solution 13. The bubbling argon and the oxygen, as well as any other volatile matter, rise to through the vapor space 39 and are eliminated from the container 11 by the outlet pipe 41. The pipe

  inlet 31 and outlet line 41 are both

  jetties tightly in a two-hole plug 43, placed in

the access pass 15.

  A battery or a DC power supply 40 is provided with a potentiometer 42 having a cursor 44 and a resistance

  46. The cursor 44 is connected by a conductor 50 to the counterelect

  trode 52. The counter electrode 52 may be made of any suitable electrode material. For most uses, an iron electrode has been found to be satisfactory. The counter-electrode 52 is tightly secured in the access neck 17, by means of the

cap 54.

  A graphite electrode 56 of low porosity and high density is secured in the access neck 20 of the container 10, by means of the plug 57, and is connected by the conductive wire 58, the ammeter 60 and the conductor 62, to the positive side of the battery 40. A reference electrode 70 such as a saturated calomel electrode is fixed through the plug 72 in an access neck 18 of the container 11 and is connected by the conductors 74 and 76 and the voltmeter 78 to conductor 58 of the graphite electrode. To start the device, it is important to rotate the graphite electrode 56 in order to ensure effective contact of the electrode with the constituents of solution 12,

  with replacement of the redox couple consumed on the surface of the electrode.

  This is obtained by means of a driving device 80 and a mechanism

  geared gear 82, shown diagrammatically in FIG. 1.

  A range of rotational speeds can 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 elect

  trode becomes more sensitive to external vibrations and less sensitive

  for the detection of low concentrations of bacteria.

  i5 To start the analysis operation, we adjust the cur-

  sor 44 of the potentiometer along the resistor 46, until oue

  the value indicated by the volmeter 78 is essentially 0.0.

  After, or at the same time, the oxygen dissolved in solution 12 is purged with argon, any remaining trace of oxygen is preferably eliminated by adding, for example, enough ferrous EDTA to reduce practically to zero current intensity

measured by the ammeter 60.

  A graph is drawn of the current intensity measured by the ammeter 60 in the analysis circuit (that is to say between the graphite electrode 56 and the counter electrode 52) as illustrated in FIG. 2. Typically the variation in current intensity over time is initially non-linear, but after a brief period, a linear relationship is established. The slope of the linear portion of the curve (indicated by the tangent in Figure 2) is proportional to the reaction speed of the redox couple at

  the electrode 56, under the effect of the catalytic action of the agent enzy-

  matic. A typical relationship between the reaction rate and the concentration of bacteria in a saturated glucose solution and

  methyLene blue (relative to the amount of bacteria available

  figures) is illustrated in Figure 3.

  This example is carried out by using a sample of

  300 ml of flood water, taken from a storm sewer or other drainage ditch

  Also, in container 10 with 0.112 g of disodium EDTA so that the concentration of disodium EDTA in the solution is 0.001 M. To this solution are added 1.633 g of KH2PO4 and 3.136 g of K2HPO4 so that the phosphate concentration of the solution is 0.1 M. The pH is adjusted to 7 using 0.5 N NaOH and to the solution is added 0.5 g of glucose (0.01 M) and 15 ml of methylene blue 0.01 M (5 x 104 M). The solution is then brought into contact with the electrodes; the rotating electrode having been previously cleaned by polishing

  with fine sandpaper, then polished on a glass plate.

  The carbon electrode has a surface area of 0.30 cm2

and rotates at 1000 rpm. -.

  The voltage is adjusted so that the rotating electrode is at 0.00 volts relative to the saturated calomel electrode. The current is measured as a function of time. When the slope of the current variation in. time function becomes constant-we determine the slope

  and we calculate the number of bacteria from -the standard curve-

  swim. In this example, the reaction rates determined are used.

  mined from The slope of the tangent of Figure 2, as shown by the broken lines in Figure 3, to establish

6 7

  that the concentration of bacteria is between 10 and 107 celtluies

  per milliliter of solution, when the reaction rate is approximately

approximately 6.25 x 109.

  You can prepare graphs similar to Figure 3 using standard concentrations of bacteria: or others

  enzymatic agents under standard test conditions. After preparation

  ration of this calibration curve, it can be used repeatedly for analyzes of different samples of solutions containing the same bacteria or enzymatic agents or bacteria or agents

similar enzymes.

  Figure 4 is a section along Lines 4-4 of the elect

  very porous and very dense graphite trode-56 illustrated by-

  Figure 1. The electrode 56 consists of a central part of

  graphite 96, which is typically a waterproof cylindrical element

  mable, very pure, having a diameter of about 0.1 cm to about 10 cm, preferably about 0.3 cm to about 1.0 cm. The graphite should have a porosity of less than about 10%. This is important for - minimizing - the absorption of the solution which could be entrained in a subsequent sample to be analyzed. Also any absorption in the interstices of the pores of an electrode has an electrical effect analogous to the increase in the surface of the electrode, that is to say increases the speed of oxidation of the redox couple and can produce readings. erroneous, in particular, for weak

  rotational speeds of the electrode.

  The coating 98 around the outer surface of the

  cylindrical rod 96 can be made of a suitable impermeable material

  any prayer. Preferably a polymeric material (such as an epoxy resin, a polyurethane, or the like) is used because the rods

  of graphite can be immersed in such materials and the coating

  Adhering can be cured relatively quickly to form a hard, dense protective layer. It is not necessary for the layer to be excessively thick, as long as it is impermeable to the liquid and acts as an insulator, preventing the passage of a significant current with respect to the conductivity of the graphite. Generally, about 1 mm thick epoxy resin coatings have been found

acceptable.

  After application of the coating 98, the terminal portion is removed to reveal an uncoated graphite surface (see FIG. 1) at the end of the electrode. The surface 110 therefore constitutes the only contact surface between the graphite and the solution 12, the protective coating 98 reaching at least the vapor space 35 of the container 10 and preferably covering the entire

  of the graphite portion of the electrode.

  - The terminal surface 10 must be ground or sanded, for example with very fine sandpaper, to remove all the scratches and form a flat surface. It is then best to polish it using a thin, hard paper on a flat, hard surface, such as a glass plate, until the entire surface has a metallic luster. The electrode is then rinsed with methanol and distilled water to remove all the oils and

  other impurities just before use.

  Thanks to the very smooth and shiny waterproof surface 110, it is possible to obtain high accuracy in the correlation of the consecutive measurements, since it is possible to obtain very reproducible electrical circuits with very precise apparent surfaces of

the graphite electrode.

  A particularly interesting characteristic of the coated graphite electrode of the invention is its use for carrying out consecutive analyzes on different samples of solution 12. For example, the solution can be changed or the electrode can be removed from the access neck 20 and use it in a different container with a different solution. Before inserting the graphite electrode 56 in a new solution, remove (for example

  by grinding) the lower terminal portion, to ensure the

  even very small quantities of solution absorbed from the previous analysis. It has been found that after grinding by about 6.35 mm or more of the bottom of the electrode, and then sanding and polishing the bottom surface, the electrode is suitable for use providing results that it is impossible to distinguish from those of a new electrode. In addition, unlike the electrodes of the prior art which use, for example, noble metals, it has been found that any deposit or any contamination of the external coating 98 of the electrode does not influence the accuracy of the analysis. It is therefore unnecessary to throw away the electrode when its outer surface

  is corroded or coated with foreign agents.

  Of course various modifications can be

  brought by a person skilled in the art to the devices or processes which come

  nent to be described only by way of nonlimiting examples without

depart from the scope of the invention.

10.

Claims (16)

  1. Method for measuring the concentration of an enzy- agent   material in a first solution (12) in which the first electrode (56) is at least partially immersed, in which a second electrode (52) is at least partially immersed in a second solution (13) which is in electrolytic communication (100 ) with the first solution, an electric potential is created between the first and the second electrode and the intensity of the current produced between said electrodes is related to the concentration of said enzymatic agent in said first solution, characterized in that one uses as said first electrode a graphite conductor   (96) having a porosity low enough to prevent absorption   notable solution of solution about 6.35 mm below its surface.   2. The method of claim I wherein said   graphite conductor has a porosity of less than about 10%.   3. Method according to claim 1 characterized in that said conductor (96) is cylindrical and rotates around its longitudinal axis at a speed sufficient to ensure its contact   effective with the constituents of said first solution.   4. Method according to claim 3 characterized in that said speed is in the range of about 100 rpm to about 2000 rpm.   5. Method according to claim 4 characterized in that the submerged portion of said cylindrical conductor (96) is coated over its entire surface, with the exception of the terminal surface   submerged, a layer of electrical insulation (98) which is impermeable able to said first solution.   6. Method according to claim 5 characterized in that said cylindrical conductor (96) has a diameter between   about 0.1 and about 10 centimeters. -   - 7. Method according to claim 6 characterized in that said conductor (96) has a diameter between about 0.3 and about 1.0 cm.   8. Method according to claim 1 characterized in that said first solution (12) consists of an aqueous solution containing an enzymatic agent, a redox couple and a substrate = 2567268   and in that it further comprises the stage of adding a sufficient amount of disodium EDTA to significantly improve the   precision of the measurement of the concentration of said enzymatic agent.   9. Method for preparing an electrode (56) intended to be at least partially submerged in a solution and for carrying out precise measurements of the intensity of the current flowing through it, characterized in that it consists in:   have a cylindrical graphite conductor (96) having a diameter   meter in the range of about 0.1 to about 10 cm and a porosity less than about 10%,   coating the lateral surface of said cylindrical conductor with a material   polymer third (98) impermeable to said solution and,   flatten, equalize and polish the terminal surface (110) of said conduc-   cylindrical to give it a metallic sheen 10. Electrode characterized in that it has been prepared   according to the method of claim 9.   11. A method for measuring the concentration of an enzyme in an aqueous solution (12) containing said enzyme and dissolved oxygen comprising: introducing a sample of said solution into a container (10), buffering said solution at an essentially neutral pH, the addition of a substrate and a redox couple to said solution,   said substrate and said redox couple being able to pass from an oxy-   dation to another under the catalytic effect of said enzyme, elimination of said dissolved oxygen from said aqueous solution after the addition of said substrate and said redox couple,   the use of an electrolytic solution (13) contained in a container   separate pient (11), the electrolytic connection (100) of said aqueous solution (12) and said electrolytic solution (13),   at least partial immersion of a counter electrode- (52) in -   said electrolytic solution, at least partially immersing a graphite electrode (56) in   said aqueous solution, said graphite electrode being constituted   killed by a cylindrical graphite conductor (96) having a diameter of between about 0.1 and about 10 centimeters and a porosity of less than about 10%, said conductor being coated on its lateral surface with an insulating coating (98) coating being impermeable to said aqueous solution and the finished surface (110) of said cylindrical conductor not comprising said coating and being flat, smooth and polished so that it has a metallic luster,   the rotation of said graphite electrode around its long axis   tudinaL at a speed of between approximately 100 and approximately 2-000 tri min with application of an electrical potential between said graphite electrode and said counterelectrode,   the measurement (60) of the intensity of the current between said counter-elect   trode (52) and said graphite electrode (56) -, and -   The determination of the concentration of the enzyme in said aqueous solution, from the rate of variation of said intensity of the current. 12. Method according to claim 11 characterized in that   that said counter electrode (52) is made of iron.   13. The method of claim 11 characterized in that said enzyme consists of bacteria and said substrate and   said coupThe redox consist of glucose and methyCene blue.   14. The method of claim 13 characterized in that-metal ions are present in said aqueous solution (1) as impurities and in that it further comprises the stage of adding a sufficient amount of disodium EDTA for minimize the interference of said metal ions which interfere with the accuracy of said enzyme measurements.   15. The method of claim 14 further comprising removing at least about 6.35 mm from the submerged end of said graphite electrode (56) after measuring the intensity of the current flowing through it; leveling, leveling and polishing the newly exposed terminal surface (110) until a metallic luster is obtained and the said reuse graphite electrode.   16. The method of claim 11 characterized in that said dissolved oxygen is removed from said aqueous solution by bubbling an inert gas containing no oxygen in this solution.