US3526578A - Method of deoxygenating solutions in making biological determinations - Google Patents

Description

H. P. SILVERMAN 3,526,578

- BIOLOGICAL DETERMINATIONS 2 Sheets-Sheet 1 INVENTOR. Hzeeser I? .571. VEEMA/V flea/m ez/yr METHOD OF DEOXYGENATING SOLUTIONS IN MAKING Sept. 1, 1970 Filed March 30, 1967 Mb W39 8 Q Sept. 1, 1970 H. P. SILVERMAN 3,526,578

METHOD OF DEOXYGENATING SOLUTIONS IN MAKING BIOLOGICAL DETERMINATIONS Filed March 30, 1967 2 Sheets-Sheet 2 m) .z N356;

& 3 8 0 Q I I I I 9 l/ R t I s 4 k I Z r g i Q r k v fi K'r I x l J 6 6 Q Q Q Q v \Q Q N NEE/(X0 2 INVENTOR. L #2795527 F 574 Vf/QMfi/V United States Patent 3,526,578 METHOD OF DEOXYGENATING SOLUTIONS IN MAKING BIOLOGICAL DETERMINATIONS Herbert P. Silverman, Orange, Calif., assignor to E. I.

du Pont de Nemours and Company, Wilmington, Del.,

a corporation of Delaware Filed Mar. 30, 1967, Ser. No. 627,090 Int. Cl. B01k 1/00; G01n 27/00 U.S. Cl. 2041 18 Claims ABSTRACT OF THE DISCLOSURE A method of deoxygenating solutions and performing bioligical determinations together with apparatus for performing the same comprising the following steps: (1) providing in an oxygen-containing solution a redox couple; (2) passing an electric current through the solution by means of electrodes immersed therein until the reduced form of the redox couple removes substantially all of the oxygen from solution; (3) introducing into the solution the reactants of an enzymatic reaction; (4) following the progress of the reaction by relating the rate of change in the current flow in a sensing circuit to a characteristic of the enzyme reaction by means of a calibration factor. The calibration factor is obtained by deoxygenating the solution and correlating the amount of the redox couple converted to the reduced form by a predetermined number of coulombs to the change in current flow in the sensing circuit.

CROSS REFERENCES The present invention is particularly related to the subject matter of co-pending patent application Ser. .No. 402,788, filed Oct. 9, 1964, for Method of Determining Microbial Populations, Enzyme Activity, and Substrate Concentrations by Electrochemical Analysis. The disclo sure of such application is hereby specifically incorporated herein and made a part hereof. It is to be understood, however, that the present invention is not limited to the effecting of deoxygenation for the purposes specified in the cited patent application, being additionally usable in other applications wherein it is desired to effect the rapid and complete removal of oxygen from various solutions, etc.

SUMMARY OF THE INVENTION The above-cited patent application describes and claims an important new method of determining microbial populations, enzyme activity, substrate concentrations, etc., in an electrochemical manner and in the substantial absence of oxygen. Specific reference is made in such patent application to an oxygen-removal step whereby a suitable scavenging gas is bubbled through the solution for a substantial period of time. It has been found, however, that such methods of scavenging oxygen from solutions prior to analysis (or prior to the carrying out of certain chemical reactions) is a time consuming and awkward procedure. In fact, the removal of oxygen is very frequently the most time consuming operation in the total method. The physical removal of oxygen, such as by the above described method and/or, for example, by the application of a vacuum, is difficult to accomplish and does not result in complete oxygen removal unless the procedure is repeated an excessive number of times. It is pointed out that although a heating step would aid in oxygen removal, the use of heat is normally contraindicated because of the instability of the material.

Other prior-art methods of deoxygenating solutions include the use of certain chemicals, for example sulfur dioxide, but there are subject to important disadvantages. Thus, for example, such chemicals are normally detrimental to the biological systems described in the cited 3,526,578 Patented Sept. 1, 1970 ICC patent application, and/or are so unstable as to make them impractical for normal usage.

Electrochemical methods of removing oxygen have also been employed but, to the best of the knowledge of applicant, such methods have involved the direct action of oxygen with an electrode. Such prior-art electrochemical methods are subject to important disadvantages, one of which is that a long time period is required for the diffusion of oxygen into contact with the electrode.

In view of the above and other factors, it is an object of the present invention to provide an electrochemical method of removing oxygen from solutions in such manner that all traces of oxygen are eliminated rapidly, easily and economically.

A further object is to provide an electrochemical deoxygenation method wherein the reagent is generated in situ, and wherein only small quantities of reagent are required.

A further object is to provide a deoxygenation method wherein no complex mechanical systems are required, the apparatus being instead simple and highly portable.

Another object is to provide a method of effecting electrochemical deoxygenation by use of a redox couple both forms of which are highly soluble in water, the reduced form of which reacts rapidly with oxygen, the oxidized form of which is reducible electrochemically in an aqueous system, which effects deoxygenation rapidly in a pH range suitable for biochemical analysis, and which is nontoxic to biological systems.

A further object is to provide a method of determining microbial populations and activities, etc., and determining enzyme activities and substrate concentrations, by first effecting a substantially complete electrochemical removal of oxygen from the solution, and thereafter effecting a biological analysis electrochemically.

An additional object is to provide a method of determining microbial populations, enzyme activities, etc., by use of two redox couples one of which is patricularly effective in removing electrochemically all traces of oxygen from the solution, and the other of which is particularly effective in the eletcrochemical determination of the rate of an enzyme-catalyzed reaction.

A further object of the invention is to provide a highly improved, simple and effective method of calibrating the electrodes employed in the instrumentation for electrochemical analysis, thereby making it possible to correlate accurately the results of electrochemical determinations made at different times and/or with different electrodes.

For a more detailed understanding of the invention, reference is made to the following description of various embodiments thereof and to the accompanying drawings in which:

FIG. 1 is a view, primarily in vertical section, schematically representing one form of apparatus for use in performing the present method; and

FIG. 2 is a graph showing the percentage of oxygen in the solution, and current flowing through the solution, as plotted against time.

In order to facilitate an understanding of the present method, there will first be described one-form of apparatus for performing the same. Thereafter, the method will be described as employed in deoxygenating solutions in general, following which there will be described the use of the method in removing oxygen prior to effecting electrochemical determinations of microbial populations, enzyme activities, etc. Finally, the method of calibrating electrodes will be set forth.

As used in the present specification and claims, the term solution includes suspensions and emulsions, and also includes relatively pure water and certain other liquids.

Referring to FIG. 1, the apparatus employed in performing the present invention may comprise two three- 3 necked half cells and 11 which are connected together by a suitable joint 12. The chambers 13 and 14 defined by the respective half cells 10 and 11 should, in order to insure against an undesired interchange of reaction products or other substances between the half cells, be separated by a suitable ion exchange membrane 15.

The membrane 15 may comprise, for example, a product of the American Machine & Foundry Company designated AMF-ion Al04 DF anion exchange membrane. The half cells 10 and 11 are preferably formed of glass because such substance effects minimum retention of gas, and because such substance permits maximum inspection of the contained solution or solutions.

Proceeding first to a description of the elements associated with the half cell 10, these include electrodes 17 and 18 which are mounted in chamber 13 by means of a rubber (or other sealing) stopper 19 in one of the three necks, and a third electrode 21 which is mounted in another one of the necks by means of a Teflon (or other suitable) stirring gland 22.

The electrode 17 is a suitable reference electrode, for example a cylindrically-shaped saturated calomel electrode (SCE), the upper end of which is sealingly inserted through an opening in the rubber stopper 19. The electrode 18 has a tubular body the diameter of which is substantially larger than that of the SCE 17, such body being mounted coaxially around the SCE by means of a wire 23 which extends sealingly through stopper 19. Electrode 18 is preferably a relatively large-area electrode, the area being, for example, 10 square centimeters. Such electrode 18 is preferably formed of expanded noble metal, such as platinum.

The remaining electrode in chamber 13, namely the electrode 21, should be a relatively small-area electrode (having an area much smaller than that of electrode 18), and may be constantly moved as by rotation. Such electrode may be termed a microelectrode or sensing electrode. In the illustrated embodiment, the microelectrode 21 is the radially-protruding cylindrical end of a platinum wire which extends through a glass rod 24, the wire being sealingly embedded in the rod. The area of the protruding electrode portion 21 may be, for example, 0.1 square centimeter.

Rod 24 is provided with an enlarged portion (or surrounded by a suitable sealing means) 26 which is rotatably and sealingly mounted in an axial bore in the aboveindicated stirring gland 22. Rod 24 and thus microelectrode 21 are preferably continuously rotated, for example by an electric motor which is schematically indicated at 27 and associated with the rod as by the drive schematically represented at 28. The speed of rotation of the microelectrode 21 should be rapid, for example 600 r.p.m.

The remaining neck in the half cell 10 contains a stopper 29 (formed of rubber or other suitable sealing substance) through which is inserted a tube 31 having at the lower end thereof a dispersive element 32. Element 32 may comprise, for example, a mass of porous glass adapted to break up into fine bubbles a gas introduced therein from tube 31 by means of a suitable gas source indicated at 33. When a valve 34 in tube 31 is in open condition, argon or other suitable oxygen-scavenging or purging gas is introduced into the liquid contained within chamber 13, to thereby provide an initial oxygen-removing step relative to the liquid and relative to the space above the liquid in cell 10.

An additional tube, numbered 36, is sealingly passed through stopper 29 and is provided at the upper end thereof with a rubber (or other suitable sealing) septum 37 through which a sample (for example, comprising an enzyme, species of bacteria, etc.) may be introduced as by means of a hypodermic needle. A third tube 38 communicates with tube 36 and is provided with a suitable valve 39 to control gas fiow therethrough. When valve 39 is open, gas introduced into the solution by means of tube 31 and dispersive element 32 may be vented to the atmosphere through tubes 36 and 38.

The elements associated with the remaining half cell, number 11, are illustrated to include a fourth electrode 41 which is suspended on a rod 42 passed sealingly through a rubber stopper 43 in one of the necks of the cell. Electrode 41 and the associated rod 42 are formed of a suitable noble metal, such as platinum. Electrode 41 should have a relatively large area, for example 10 square centimeters as described above relative to the electrode 18 in cell 10. Such relatively large-area electrodes 18 and 41 permit passage of substantial currents through the solution, at relatively low voltages.

The remaining two necks of half cell 11 contain, respectively, and in suitable sealing stoppers 44 and 45, a gas-introduction tube 46 and a vent tube 47. Tube 46 has a dispersive element 48 at the lower end thereof, and connects through a suitable valve 49 with a source 50 of argon or other suitable oxygen-scavenging gas. The vent tube 47 communicates through a valve 51 with the ambient atmosphere. Thus, gas may be introduced from source 50 through valve 49, tube 46 and dispersive element 48 into the solution contained in chamber 14, following which such gas is vented through tube 47 and valve 51.

It is pointed out that the dispersive elements 32 and 48 are disposed at much lower elevations than are the lower ends of vent tubes 36 and 47, in order that such vent tubes may communicate with portions of the half cells which are above the contained liquids.

Stirring bars 52 and 53 are disposed on the bottoms of the half cells 10 and 11 and are formed of magnetizable material. Accordingly, and when such bars are magnetically associated with suitable magnetic stirrers 54 and 55, the bars 52 and 53 are suitably rotated or otherwise moved in order to effect continuous stirring of the liquids in the cells.

Proceeding next to a description of one form of electric circuit means for interrelating different pairs of the four electrodes 17, 18, 21 and 41, this is illustrated schematically to comprise a DC. source 61 having the positive terminal thereof connected through a lead 62 to rod 42. The negative terminal of source 61 is connected through a suitable switch 63 to the wire 23 which supports electrode 18. Thus, when switch 63 is closed, an electric circuit is provided from source 61 through the large-area electrodes 18 and 41 and through the liquids in which such electrodes are immersed as will be set forth hereinafter. The current flow through the described circuit is normally in the milliampere range, and performs functions such as electrochemical deoxygenation. At other times, the indicated current performs functions such as converting a predetermined quantity of oxidized mediator or redox couple to the reduced form thereof. A suitable ammeter 64 is interposed in lead 62 in order to measure the current in the described circuit.

The remaining electrodes 17 and 21 are also connected in a circuit, which may be designated the sensing circuit. This is effected by means of a lead 65 which extends from the SCE 17 to one terminal of a very sensitive ammeter 66. The remaining terminal of such ammeter is connected through a lead 67 to the wire which extends through glass rod 24 to the microelectrode 21. Thus, electrodes 17 and 21 are associated with each other through a circuit which includes the liquid (in chamber 13) in which such electrodes are immersed. A sensitive voltmeter 68 is connected between leads 65 and 67 in order to measure the potential difference between electrodes 17 and 21.

Ammeter 64 may be a milliammeter, whereas ammeter 66 may be a microammeter having an extended (more sensitive) scale. Voltmeter 68 is an extremely highresistance voltmeter, for example of a vacuum-tube variety, adapted to measure the potential difference without altering substantially the current fiow through ammeter 66.

The resistance in the circuit between electrodes 17 and 21 is preferably so regulated that the potential of the microelectrode 21 is in the vicinity of zero volts vs. the SCE 17.

GENERAL DESCRIPTION OF THE METHOD OF ELECTROCHEMICAL DEOXYGENATION Stated generally, the method of electrochemical deoxygenation comprises providing in a suitable liquid medium the oxidized form of a redox couple, and passing through such medium sufficient current to reduce at least a substantial proportion of the redox couple and thereby cause the reduced redox couple to react with and thus remove the oxygen in the medium. The electrodes (such as 18 and 41) between which the current is passed are caused to have sufiicient surface areas that low current densities may be employed while still maintaining a current sufficiently large to remove the oxygen at a rapid rate. It is emphasized that, as previously indicated, the present method achieves the important advantage, in comparison with the direct reduction of oxygen, that it is not dependent upon the diffusion of oxygen to the electrode.

The method requires the use of a redox couple having particular characteristics relative to solubility and other factors, the characteristics being such that oxygen may be removed in a rapid and practical manner. Stated more definitely, the electroactive material employed for the electrochemical deoxygenation should have the following characteristics:

(1) The oxidized form should be reducible electrochemically in an aqueous system.

(2) The reduced form should react rapidly with oxygen. (3) Both forms of the material should be soluble in water.

Furthermore, if biological or other testing is to be effected after oxygen removal, the redox couple should, in addition, have the following characteristics:

1) Both forms should be soluble in the pH range wherein the desired tests are to be made (the preferred pH range for bacteria being on the order of about 5 to about 9).

(2) Both forms should not be toxic to or otherwise interfere with the biological or other systems to be tested subsequent to oxygen removal.

It might be supposed that a substrate such as methylene blue, the action of which was described at length in the above-cited patent application, would be suitable for electrochemical deoxygenation. It has been found, however, that such substance is not practical to employ for the present purpose, one reason being that it builds up upon the electrode a resistive barrier Which greatly reduces current flow through the solution and which makes even substantially complete deoxygenation an excessively long and impractical procedure.

Applicant has found that a metallic ion complex and more specifically a metallic chelate, which meets the above-identicated requirements, is highly and surprisingly satisfactory for effecting electrochemical deoxygenation. The following metallic ion redox couples, inter alia, either of themselves or with appropriate complexing agents, may be incorporated in the subject deoxygenation method:

Any complexing agent (ligand) compatible with the requisite properties of the redox couple can be employed, e.g., anion of EDTA (ethylenediamine tetraacetic acid), anion of an amino acid, etc. Suitable metallic ion complexes include, among others, cuperic-amino acid and metallic chelates, preferably, ferric-EDTA.

It is to be pointed out that the embodiment of ferric- EDTA in the attendant description is merely exemplary of redox couples satisfactory for accomplishing the stated objects of the invention.

One way of preparing a ferric-EDTA (using a 0.01 M solution as an example) is as follows: g. of disodium ethylenediamine tetraacetic acid (EDTA) were dissolved in 800 ml. of water, 4.82 g. (0.01 mole) of ferric ammonium sulfate were added, and the pH was adjusted to 7 by dropwise addition of concentrated ammonium hydroxide. One hundred ml. of 1 M sodium phosphate pH 7 were added and the solution wasbrought to 1 liter with distilled water. The resulting ferric-EDTA in a buffered solution was stored in a red bottle to prevent decomposition by light.

The solutions to be deoxygenated electrochemically are caused to contain the ferric-EDTA, either alone or in combination with a biological mediator such as methylene blue (as will be set forth hereinafter). The ferric-EDTA has been employed in concentrations ranging from 5 10 M to 1X10 M. The higher concentrations have been employed in the absence of any buffer (such as phosphate buffer), but at least relative to the lower concentrations it is preferred, to employ a suitable buffer. The described concentrations are not to be regarded as limiting the scope of the present patent application.

Highly rapid and successful oxygen removal has been demonstrated, utilizing the ferric-EDTA, both in the presence and absence of glucose, bacteria, enzymes, etc. It is possible to employ the electrochemical deoxygenation method to remove allof the oxygen from a saturated solution, or to use the method for removal of oxygen traces present after the solution has been purged with argon or other suitable scavenging gas.

It is strongly emphasized that, for many purposes such as in the measuring of low levels (10 cells/ml.) of bacteria, even small traces of residual oxygen interfere and must be completely eliminated. Furthermore, as will be stated below, even small traces of oxygen in the solution prevent achievement of satisfactory electrode calibration. Therefore, and additionally because no special gasscrubbing equipment is required, the present method of electrochemical deoxygenation has been found to be highly important and satisfactory in many testing and other procedures. Not only are the last traces of oxygen rapidly removed, but the required amount of ferric-EDTA is so small that there is substantially no chance of poisoning the system. The above-indicated generation (and regeneration) of the reagent (ferric-EDTA) in situ permits the use of labile reducing agents.

With particular reference to the apparatus shown in FIG. 1, the method of electrochemical deoxygenation may be performed in the following manner. The first step comprises providing a conductive liquid medium which is suitable for performance of the test after deoxygenation has been completed. For example, relative to the use of the glucose-glucose oXidase-methylene blue mixture for performing biological testing as described in the cited patent application, the above-indicated phosphate buffer adjusted to pH of 7.0 has been found to be highly satisfactory. Other buffers, and other pHs, may be employed if desired. As an illustration, effective buffering action is achieved by preparing a phosphate buffer as follows: Stock solutions of (a) 0.1 M potassium dihydrogen phosphate (KH PO4), and of (b) 0.1 M di-potassium hydrogen phos hate (K HPO are made up. These two solutions may be mixed in different proportions to give solutions buffered at different pH values. A pH of 7 is obtainedby mixing in the ratio of four parts of solution (a) to six parts of solution (b), by volume. The described buffer solution is one form of a compatible conductive (electrically) medium. The word compatible is intended to denote an absence of poisoning of or harmful effects upon enzyme activity, etc.

The particular circumstances. must be such that the ferric-EDTA does not precipitate. If precipitation occurs,

7 a different buffer concentration or another buffer (such as an acetate) is employed.

The described liquid medium is introduced into chambers 13 and 14, the liquid surfaces being indicated at 71 and 72 and being disposed between the electrodes and the vent openings through tubes 36 and 47. Thus, the electrodes are immersed in the solution.

It is pointed out that the same liquid need not be provided in each of the chambers 13 and 14. Furthermore, as will be stated hereinafter, it is common to provide in the chamber 13 substances additional to (or other than) the substances present in chamber 14. Thus, for example, methylene blue may be added to chamber 13.

It may be desirable to provide a suitable oxidizable substance in chamber 14 in order to prevent electrolysis of the water. For example, ferrous ion and a suitable chloride salt may be employed in place of the phosphate buffer. It is emphasized that the use of such substances in chamber 14 is not essential, particularly if argon is bubbled continuously through the solution in chamber 14 during continuance of the method.

The next step in the method normally comprises opening both gas valves 34 and 49, and both vent valves 39 and 51, so that argon or other suitable scrubbing gas is introduced into the solution through the dispersive elements 32 and 48. Such argon introduction is preferably continued until a large percentage of the oxygen has been removed from the liquid, and until all air has been removed from the air space above liquid surfaces 71 and 72. Thereafter, the various valves 34, 39, 49 and 51 may be closed to seal the system from the gas sources and from the atmosphere. Alternatively, tubes 31 and 46 may be raised until the elements 32 and 48 are disposed above surfaces 71 and 72, the argon fiow then being changed from through the solutions to over such solutions. In this manner, a positive pressure is maintained in the half cells 10 and 11 to insure against any leakage of oxygen into such cells.

It is pointed out that the membrane prevents interchange of various substances between chambers 13 and 14, while at the same time permitting flow of current therethrough. However, the membrane 15 may not effectively prevent diffusion of oxygen between the chambers, thus making it desirable to remove the oxygen from chamber 14 by the method indicated above.

As previously stated, it is not necessary to employ any gas-scrubbing step, particularly if the cells are sufficiently full to insure against the presence of any air in the cells above liquid surfaces 71 and 72.

Thereafter, or prior to the described introduction of argon into the cells, a suitable amount of the ferric- EDTA solution is mixed with the described conductive medium in cell 10. As an example, the ferric-EDTA may be present in 0.01 M concentration.

Because of the presence of at least small amounts of oxygen in the solution, the current flowing in the sensing circuit comprising microelectrode 21 and SCE 17 will be cathodic (electron flow through the metallic components being in a direction from electrode 17 to electrode 21).

The next step in the method comprises closing of switch 63 to effect flow of current through the solutions and through membrane 15 between large-area electrodes 18 and 41. DC. source 61 preferably comprises a substantially constant-current device known as an amperostat, the current flow being maintained relatively constant (for example between about 20 milliamperes and about milliamperes). Because of the indicated polarity of the source 61, the direction of electron flow through the metallic circuit is from electrode 41 to electrode 18.

The result of the described current flow from source 61 is that the ferric-EDTA is converted, at the cathode 18, to ferrous-EDTA. The ferrous-EDTA thus generated reacts rapidly with the residual oxygen present in the solution, the result being that the current flow through the sensing circuit 17-21 becomes progressively less cathodic. Such sensing current flow is indicated by the highly sensitive ammeter 66, the voltage being preferably maintained close to 0.0 volt vs. SCE as indicated by voltmeter 68. A preferred voltage range is between --0.1 and +0.1 volt vs. SCE.

The magnetic stirrers 54 and 5-5 are maintained continuously in operation, to produce constant agitation of the solutions.

The switch 63 is maintained closed until the current flow in the sensing circuit 17-21 becomes slightly anodic (electron flow direction being through the metallic circuit from 21 to 17). It is then known that all residual oxygen has been removed from the liquid in cell 10. A desired testing procedure may then be performed in cell 10 as will be set forth in detail hereinafter.

Referring next to FIG. 2, there is shown a graph illustrating the rapid removal of oxygen from the solution in cell 10 by the present electrochemical method. The solid-line curve 73 (and corresponding solid dots) represents the amount of oxygen present in the solution in chamber 13, measured as a percent of saturation, such percent being plotted against time in minutes. Because the curve 73 starts at it is pointed out that the indicated curve is one representing a situation wherein there was no preliminary purging step by means of argon or other gas. A second solid-line curve 74 shown in FIG. 2 (and the corresponding solid dots) represents the current as measured by ammeter 64.

Curve 73 indicates that the amount of oxygen was changed from 100% to 0% in a time period on the order of 12 minutes. Curve 74 indicates that the current flowing from source 61 through the electrodes 18 and 41 remained relatively constant, between about 20 milliamperes and about 25 milliamperes.

The indicated fast and simple procedure is to be compared with prior-art procedures, wherein residual oxygen was almost always present despite scavenging operations which required time periods measured in hours instead of minutes.

METHOD OF MAKING BIOLOGICAL DETER- MINATIONS BY THE PRESENT METHOD It has been found that ferric-EDTA is reduced relatively slowly by various species of bacteria, enzymes, etc., which are the subject of biological determinations as set forth in the cited patent application. However, it has also been discovered that mixtures of ferric-EDTA with certain mediators (redox couples) set forth in the cited patent application produce highly satisfactory results relative to both electrochemical deoxygenation and biological sensing and testing. For example, it was discovered that a mixture of ferric-EDTA and methylene blue could be deoxygenated chemically in a short period of time, approximately 10 minutes, and then employed for the detection of microorganisms such as Escherichia coli (E. coli). Mixtures of ferric-EDTA with other mediators, such as phenazine methosulphate, are also readily deoxygenated and produce excellent results in performing biological testing.

Referring again to FIG. 2, there is indicated at 76 a dashed-line curve which corresponds to the aboveoxygenation was affected relative to a mixture of ferricdescribed curve 73 except that the electrochemical de- EDTA and methylene blue. More specifically, the mixture tested comprised 10- M methylene blue and 0.01 M ferric-EDTA. Such curve, which extends between points represented by small circles, shows the complete deoxygenation was achieved in the above-mentioned time period on the order of 10 minutes instead of about 12 minutes as was the case relative to curve 73 wherein no methylene blue was present.

The current employed to effect the electrochemical deoxygenation is indicated by the dashed-line curve 77 at the top of FIG. 2, and shows that the current range (as measured by ammeter 64) was again generally between about 20 milliamperes and about 25 milliamperes.

Once electrochemical deoxygenation has been completed, as set forth in detail above, it is merely necessary to inject, by means of a hypodermic needle, a test sample through the septum 37 into chamber 13. This permits the injection to be performed without introducing oxygen. The test sample may comprise any of the numerous substances set forth in the cited patent application, for example whole, live cells of E. coli, an enzyme such as glucose oxidase, or yeast (Saccharomyces carlsbergensis), etc. Switch 63 may be opened, to discontinue How of current between the large-area electrodes, before the sample is introduced.

Assuming that there was initially present in (or subsequently introduced into) the chamber 13 the substrate (such as glucose) necessary for an enzyme-catalyzed electrochemical reaction described in detail in the cited patent application, the injected sample initiates the reaction which then produces a change in the current indicated by the sensitive ammeter 66. Thus, the enzymatic reaction causes the current flow in the circuit comprising electrodes 17 and 21 to again become cathodic, and then become progressively more cathodic. As described in detail in the cited application, the rate of change in the current measured by ammeter 66 is an accurate indication of a biological characteristic (such as activity or numbers of the microorganism, effectiveness of the enzyme, etc.) to be determined by the present method. The rate of change of such current is an indication of the rate of the enzymatic reaction.

It is to be understood that, if desired, an external source of current may be employed during the biological sensing procedure, as described in the cited application. The procedure may be of the amperometric type, amperostatic type, etc., as therein described. It will therefore be understood that the combination of electrochemical deoxygenation and electrochemical biological sensing described in the present application and in the above-cited application produces, in a very short time period such as 15 or 20 minutes, a highly accurate determination which is devoid of error resulting from the presence of oxygen.

It is to be understood that the order of introduction of the various reactants may be varied in certain ways, without departing from the scope of. the present invention. For example, it is possible to provide a large quantity of undiluted sample (such as an enzyme solution) in chamber 13, together with a compatible conductive medium such as the above-specified phosphate buffer. Ferric-EDTA is then added and the electrochemical deoxygenation procedure is performed as set forth above. After the reading of ammeter 66 has indicated that deoxygenation has been completed, methylene blue (or other desired mediator or redox couple) is added in solid form. Because the mediator is added in solid form, no oxygen is present therein, it being understood that the solid is added in such manner that no oxygen is introduced into the liquid medium. The procedure described in the present paragraph produces the advantage that there is less dilution of the sample, the reason being that a large volume of sample is initially present.

The following is a specific example: A sample of solution containing 3 10 live, whole cells bacteria/ml. of E. coli was introduced into compartment 13. Into compartment 14 a solution containing ferrous chloride was introduced. Compartment 13 was made .01 M in ferric- EDTA and the electrochemical deoxygenation was carried out by passing 20 milliamperes between electrodes 18 and 41 for approximately minutes, or until the diffusion current, as measured by electrode 21 and ammeter 66, became cathodic. Methylene blue+glucose was added in solid form to compartment 14 so that final concentrations were 10- M and 1() M, respectively. The rate of change of diffusion current with time was measured. A rate of about 9 10 milliamperes per minute was observed. The electrode was calibrated by passing a current of 50.0 microamperes for 1.00 minute and measuring the current displacement.

METHOD OF ELECTRODE CALIBRATION Under otherwise identical conditions, the diffusion current at a given electrode is proportional to the surface area of the electrode. Since the true surface area of an electrode is not easily measured, nor is it necessarily related in any specific manner to the geometric area, a satisfactory method of calibrating electrodes is necessary if results with different electrodes (and with the same electrode at different times) are to be correctly compared.

In accordance with the present calibration method, all oxygen is first removed from the solution. Thereafter, a measurement is made of the change in diffusion current resulting from the generation of a known amount of reduced electroactive material. It is emphasized that removal of oxygen is an essential step if accurate results are to be achieved, because the presence of different amounts of oxygen during different testing procedures produces a very material error. Stated otherwise, the pres ence of oxygen prevents achievement of Faradaic efficiency at an electrode such as microelectrode 21.

With reference to the apparatus shown in FIG. 1, the electrode calibration method comprises providing ferric- EDTA in the conductive liquid medium (such as the above-specified phosphate buffer) present in chamber 13. Switch 63 is then closed to effect the above-described electrochemical deoxygenation procedure, the procedure being continued until the reading of ammeter 66 indicates that all residual oxygen has been removed (it being understood that the described preliminary purging with argon may also be performed). Switch 63 is then opened, and the reading of ammeter 66 is noted. Switch 63 is then closed in order to pass through the solutions and through membrane 15 a predetermined constant current for a predetermined time period. Switch 63 is then opened.

The amount of coulombs passed through the solution being thus known, Faradays law is employed to determine the quantity of ferric-EDTA which was electrochemically converted to ferrous-EDTA. The reading of ammeter 66 is again noted, and the change in diffusion current between electrodes 17 and 21, which change is proportional to the described conversion of ferric-EDTA to ferrous-EDTA, is thus determined. The change in current is then divided by the change in concentration of ferrous-EDTA to produce a calibration factor.

The calibration factor being thus known, the apparatus is then employed to determine, for example, the population of bacteria in a particular sample. The instrument reading is then multiplied by the calibration factor to produce the true result.

To test the above calibration procedure, the entire above-described procedure is then repeated, with the same specimen but with a microelectrode different in size than the above-specified microelectrode 21. Thus, a second calibration factor is determined, following which a second determination is made of the microorganism population. The apparent result of such second determination of microorganism population is then multiplied by the ratio of the first calibration factor to the second calibration factor. Because of the complete elimination of oxygen, the result of such multiplication is (assuming all other factors are equal) substantially the result of the first-described determination of microorganism population. Substantial correlation of the test results is thus indicated.

Because of the present method, it is possible to be sure that apparent changes in the results of biological testing procedures are actually the result of changes in the samples, instead of being the result of changes in the true electrode area.

The electrodes may be solid noble metals, such as platinum, or may be suitably platinized, etc.

The appended claims should not be regarded as limited relative to sequence of steps (unless sequence is specifically stated, as by use in a claim of the word thereafter).

The foregoing detailed description is to be clearly understood as given by way of illustration and example only, the spirit and scope of this invention being limited solely by the appended claims.

I claim:

1. In a method of making a biological determination, the steps of:

providing in an electrically conductive fluid medium the oxidized form of a redox couple,

said couple having a reduced form which reacts rapidly with oxygen in said medium to remove the same, passing through said medium from an external power source sufficient electric current to convert to said reduced form. a sufiicient quantity of said redox couple to remove the oxygen from said medium, and

utilizing said medium in a testing procedure relating to the making of a biological determination.

2. A method of making biological determinations, which comprises:

providing in an electrically conductive fluid medium the oxidized form of a redox couple,

said couple having a reduced form which reacts rapidly with oxygen in said medium to remove the same,

passing through said medium from an external power source sufficient electric current to convert to said reduced from a sufficient quantity of said redox couple to remove the oxygen from said medium, providing in said medium a biological sample,

effecting an enzyme-catalyzed reaction relative to said sample, and determining electrochemically a factor relative to said enzyme-catalyzed reaction.

3. An electrochemical method of making biological determinations, which comprises:

providing in an electrically conductive fiuid medium the oxidized form of a first redox couple,

said first couple having a reduced form which reacts rapidly with oxygen in said medium to remove the same, providing in said medium at least one of the reactants of an enzyme-catalyzed reaction between a second redox couple, a substrate, and an enzyme, passing through said medium from an external power source sufficient electric current to convert to said reduced form a suflicient quantity of said first redox couple to remove the oxygen from said medium,

thereafter introducing into said medium at least one of the remaining reactants of said enzyme-catalyzed reaction to thereby initiate said reaction, and electrochemically determining the rate of said enzymecatalyzed reaction.

4. The invention as claimed in claim 3, in which said last-named step includes measuring the rate of change of current flow through said medium between electrodes immersed therein and independent of said power source.

5. The invention as claimed in claim 3, in which said first redox couple is a metallic chelate, and said second redox couple is selected from a group consisting of methylene blue and phenazine methosulphate.

6. The invention as claimed in claim 5, in which said metallic chelate is ferric-EDTA.

7. A method of effecting biological testing, which comprises:

providing in at least one sealed chamber first and second relatively large-area electrodes,

providing in said chamber a reference electrode and a relatively small-area sensing electrode, introducing into said chamber a conductive aqueous medium containing oxygen, providing in said cham- 12 her a redox couple which is rapidly reducible electrochemically in said aqueous medium, which in its reduced form reacts rapidly with oxygen, and both forms of which are highly soluble in said aqueous medium,

passing an electric current from an external source and between said large-area electrodes in such manner that said redox couple in said chamber is reduced electrochemically and reacts with the oxygen in said aqueous medium to remove the same,

electrically connecting to each other said reference electrode and said sensing electrode,

continuing said passage of current between said largearea electrodes at least until the flow of current in the circuit including said reference and sensing electrodes is substantially zero,

introducing into said aqueous medium the reactants of an enzymatic reaction, and determining the rate of change in the current flow in said circuit including said reference and sensing electrodes to thereby follow the progress of the enzymatic reaction between said reactants.

8. The invention as claimed in claim 7, in which said reactants of said enzymatic reaction include a second redox couple, a substrate and an enzyme.

9. The invention as claimed in claim 8, in which said enzyme is provided by introducing microorganisms into said medium.

10. The invention as claimed in claim 9, in which said microorganisms are whole live cells.

11. The invention as claimed in claim 7, in which said redox couple is ferric-EDTA, and in which a second redox couple is provided that is selected from a group consisting of methylene blue and phenazine methosulphate.

12. The invention as claimed in claim 7, in which said method includes passing a substantially constant current between said relatively large-area electrodes during said deoxygenation step.

13. In a method of electrochemical testing, the steps of providing two electrodes immersed in a conductive aqueous medium,

providing in said aqueous medium the oxidized form of a redox couple having a reduced form which reacts rapidly with oxygen to remove the same, passing through said medium suflicient electricity to result in deoxygenation thereof by said reduced form,

providing the oxidized form of a redox couple in said medium and passing a predetermined number of coulombs of electricity from an external source through said medium, and

correlating the amount of said redox couple converted to the reduced form thereof by said predetermined number of coulombs to the change in curent flow through said medium between said electrodes resulting from said conversion of said redox couple to the reduced form thereof, thereby providing a calibration factor. V 14. In a method of biological testing, the steps of:

providing first and second electrodes in a conductive liquid medium,

electrochemically deoxygenating said medium by electrochemically converting a redox couple to the deoxygenating reduced form thereof,

providing a redox couple in said medium,

passing a predetermined number of coulombs of electricity through said medium to thereby convert a predetermined amount of said redox couple to the reduced form thereof, determining the change in current flow between said electrodes resulting from said conversion of said redox couple to the reduced form thereof in response to flow of said predetermined number of coulombs, and

correlating said change in current flow to the amount of converted redox couple to thereby produce a calibration factor.

13 14 15. A method of biological testing, which comprises: said enzymatic reaction is between reactants including a removing the oxygen from a conductive medium by substrate, a redox couple and microorganisms.

providing a redox couple therein and passing current therethrough to thus convert electrochemically said References Cited redox couple to the deoxygenating reduced form 5 UNITED STATES PATENTS thereof, and thereafter electrochemically determin- 3 7. 1/1966 Okun et 1 204 195 ing the rate of an enzymatic reaction in said deoxy- 7 :7 5 9/1966 Garst 204. 1 1

s d p 3,328,277 6/1967 Solomons et a1 204-195 16. The inventlon as clalmed in claim 15, in whlch 3 367,849 2/1968 Blaedel et a1 204 1.1

said redox couple is a metallic chelate, and said enzymatic 10 9/1968 Rohrback et 2Q4 1 1 reaction is between reactants including a substrate, a ren dox couple and microorganisms. TA HSUNG TUNG, Primary Examiner 17. The invention as claimed in claim 15, in which said redox couple is ferric-\EDTA. 18. The invention as claimed in claim 15, in which 15 204130,195, 265, 266; 55-459 a- 3 UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 2 5 ,57 Dated September 1, 1970 lnventofls) Herbert P. Silver'man It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 11, line 33, change "from" to ---form-.

Column 12 line 53, change "curent" to --current--.

FEB 9 197?] W E- SGHUYLER, JR Hu M Gomluiom of Patents

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