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Biological analyzer and method

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US3919051A
US3919051A US48770974A US3919051A US 3919051 A US3919051 A US 3919051A US 48770974 A US48770974 A US 48770974A US 3919051 A US3919051 A US 3919051A
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solution
membrane
oxygen
flow
enzyme
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Robert B Koch
Leroy D Nelson
Gerald D Rork
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Honeywell Inc
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Honeywell Inc
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483Physical analysis of biological material
    • G01N33/487Physical analysis of biological material of liquid biological material
    • G01N33/49Blood
    • G01N33/4915Blood using flow cells

Abstract

A continuous flow method and apparatus for the determination of glucose in a biological fluid by the oxygen depletion method is provided. The biological fluid sample, along with a carrier solution, are combined and introduced into a chamber containing gel-immobilized glucose oxidase. The mixture is caused to flow sequentially through the immobilized glucose oxidase and a connecting passage the outlet of which is disposed such that the fluid impinges on an oxygen-permeable membrane where the oxygen diffusing therethrough drains sensed electrochemically. The effluent solution drians and the system is regenerated by flushing with a quantity of the reagent solution. The glucose oxidase is immobilized in a manner which allows many tests to be run without any significant loss of enzyme thereby accomplishing a considerable cost reduction in the tests.

Description

United States Patent Koch et al.

ACETATE BUFFER SUPPLY RESERVOIR CONSTANT HEAD lll] 3,919,051

Nov. 11,1975

l 5 1 ABSTRACT A continuous flow method and apparatus for the de termination of glucose in a biological fluid h the ox gen depletion method is provided. The biological fluid sample. along with a carrier solution. are combined and introduced into a chamber containing gelimmobilized glucose oxidase. The mixture is caused to Hon sequentially through the immobilized glucose oxidase and a connecting passage the outlet of which is disposed such that the fluid impinges on an o\ \gcnpermeable membrane where the oxygen dillusing therethrough drains sensed electrochemically The ctfluent solution drians and the system is regenerated b Flushing with a quantity of the reagent solution. The glucose oxidase is immobilized in a manner which al lows many tests to be run without an significant loss of em} me thereby accomplishing a considerable cost reduction in the tests.

ll Claims. 2 Drawing Figures OXYGEN ANALYZER DISPLAY DEVICE US. Patent Nov. 11, 1975 Sheet 1 of 2 3,919,051

ACETATE BUFFER SUPPLY RESERVOIR r33 I w 32 CONSTANT OXYGEN HEAD SUPPLY ANALYZER DISPLAY DEVICE FIG.I

US. Patent Nov. 11, 1975 Sheet 2 of 2 BIOLOGICAL ANALYZER AND METHOD BACKGROUND OF THE INVENTION l. Field of the Invention The present invention relates to the field of testing samples of biological fluids for specific components and, in particular, the determination of glucose in such fluids as blood by the oxygen-depletion method.

2. Description of the Prior Art The use of enzymes in the determination of various components in biological fluids is gaining rapidly as an analytical technique. This technique generally involves the measurement of the concentration of one or more products, the depletion of one or more reactants or the change in solution condition following an enzyme-catalyzed reaction in which a specific enzyme is utilized to catalyze a known reaction. An example of this is found in the determination of glucose in blood, urine or other biological fluids. In the presence of the enzyme glucose oxidase (G) the glucose in the biological fluid reacts with oxygen in the following manner:

Glucose+O H O i'kGluconic acid H O When this reaction takes place with an excess of oxygen over that required to oxidize all the glucose in the biological sample, there is a linear relationship between the depletion of oxygen and the glucose concentration in the biological sample. Thus, if the oxygen tension is measured after the reaction and compared with a known or measured value before the reaction the exact amount of glucose in the biological cample may be readily determined.

One prior art device for measuring oxygen depletion due to the above reaction is illustrated and described in US. Pat. No. 3,542,662. That reference discloses an electrochemical transducer in the form of an oxygen measuring electrode in combination with a polyacrylamide gel covering which contains an amount of immobilized glucose oxidase therein. The electrode is then immersed in a solution containing the biological fluid to be analyzed wherein the reactants diffuse into the gel matrix where the catalyzed reaction takes place and the oxygen tension is then measured through the membrane electrochemically.

It is also known in the prior art that it is advantageous to flow a solution in which a dissolved gas such as oxygen is to be measured past the membrane-covered electrode to increase the sensitivity and response of the electrode to the dissolved gas. Such a technique is described in an article by John T. Penniston An Extremely Rapid Burst of Respiration upon Aeration of Mitochondria: Measurement of Oxygen Tension with lOmsec Time Resolution"; Archives of Biochemistry and Biophysics I50, 556565 (1972).

Although the prior art devices have utilized techniques which have advanced the state of enzymatic analysis, there has long been a need for more efficient and automated systems. The enzyme electrode method is essentially a batch-type operation wherein residual products of one reaction may interfere with readings taken for subsequent tests unless special cleaning steps are utilized to remove any unreacted components and products of a previous reaction before the next test is made. Also, an additional membrane normally must be placed over the immobilized enzyme gel to prevent loss of the enzyme from the gel into the reacting solution.

SUMMARY OF THE INVENTION According to the present invention. there is provided a method and apparatus for chemical analysis which combines an improved enzyme-catalyzed reaction system with an improved oxygen sensing technique in a flow-through analytical system. By means of the pres ent invention a large number ofdeterminations may be made without replacing either the enzyme or utilizing a special cleaning procedure for the system,

Briefly, the present invention contemplates a method and apparatus for determining the concentration of a component in a biological sample. ie the determination of glucose in a fluid such as blood serum. wherein the measured sample is combined with an amount of carrier solution and introduced into a flow-through reaction chamber such as a column. for example. containing an amount of immobilized glucose oxidase where the desired reaction is catalyzed and takes place. The solution which is then substantially completely re acted then continues to flow. while still in the closed system. and is caused to impinge on a gas-permeable membrane opposite a measuring electrode in a manner which facilitates oxygen diffusion therethrough. and at the same time. by washing thereover continuously cleanses the membrane. The effluent containing the reacted sample is then discarded via a drain. The oxygen tension is measured electrochemically through the membrane during the time which the reacted sample impinges on the opposite side of the membrane allowing the dissolved oxygen to diffuse thercthrough. The system is then cleansed by allowing an additional amount ofthe reagent solution to flow through the system returning the oxygen reading to a stable base line.

The flowthrough system of the present invention may be one utilizing a constant head type pump or one operated by gravity. The flow rate may be conveniently controlled by selection of the size and length of the conduit and column components of the flow system such that the sample is completely reacted by the time the oxygen is measured at the electrode and assuring that sufficient oxygen-containing carrier solution is supplied to completely react the glucose in each sample. Thus, no built in delays or other complicated electronics are required to measure the output of the system of the invention to allow for the time ofreaction or other variable as is required in batch wise systems in which the measurement is made in the same chamber as the reaction.

Of course. if desired, a point other than the end point of the reaction may be selected and the flow standardized to obtain reproducible readings at that point.

If required the temperature of the system may be controlled as the rate of the enzyme-catalyzed reaction is somewhat temperature dependent.

BRIEF DESCRIPTION OF THE DRAWINGS In the drawings, wherein like numbers are used to denote like parts throughout the same:

FIG. I is an illustration, partially in diagramatic form and partially in section. depicting the apparatus of the invention generally and FIG. 2 is an enlarged view ofa portion of FIG. I illustrating the measurement system of the preferred embodiment.

DESCRIPTION OF THE PREFERRED EMBODIMENT FIG. I is a general. overall illustration ofthe apparatus of the invention. For purposes of clarity some ofthe figure is shown in section. and for simplification. a portion is illustrated in block form. A carrier solution in which the reaction takes place and also which serves as a cleansing solution for the entire system is supplied from a supply reservoir to a constant head supply tank ll by a conventional method such as pumping into the constant head supply tank or by overflow from a separate weir system in a well known manner. The carrier solution is supplied from the constant head supply to the remainder of the flow system of the apparatus as by a conduit 12 having a flow shutoff valve 13.

A measured sample of biological fluid to be analyzed is introduced into the system from a separate injection port 14 normally from a graduated syringe. A septum membrane 16 serves as a closure member for the bottom of the injection port. The sample then is introduced along with the carrier solution at 17 just above a reaction column 18.

One advantage of the instrument of the present invention lies in the fact that very small samples may be used in the analysis. A typical sample size is about 50 microliters. During the duration of operating the instrumerit. the shutoff valve 13 may be left continuously in the open position. The flow ofcarrier solution required to be maintained in the system for the consecutive analysis of 50 microliter samples is only about 1.0 milliliter per minute and such continuous flow will not consume significant quantities of the carrier solution. Of course. the volumes of the rest of the system components are sized commensurate with this flowrate.

The combined or test solution then passes through a porous plug 19, which may be made ofa glass frit material or may be a fine metal screen or approximately 100 mesh. The reaction column [8 contains an amount of enzyme immobilized in a finely divided gel matrix. A further porous plug 20 much like the plug 19 closes the bottom of the column and. while allowing free flow of the test solution therethrough prevents any loss of the enzyme-containing gel from the column. The test solution flows from the bottom of the column through another conduit 21 to a chamber 22 where the final oxygen content of the solution is measured. The effluent is continually discharged through a drain 23.

The conduit 21 is sized and the downstream terminal opening thereof is disposed such that the test solution enters the chamber 22 in a manner which causes it to impinge upon a membrane 24 at an angle and to strike the membrane with a relatively high velocity. The test solution. at the same time. circulates about and cleanses the chamber prior to flowing out the drain 23. This configuration is discussed in greater detail in relation to FIG. 2, below.

The membrane 24 is disposed across the bottom of an electrochemical cell 25 containing an oxygen sensitive electrode 26 and defines a barrier separating the internal cell electrolyte from the external environment of chamber 22. A liquid-tight seal is provided about the perphery of the interface between the electrochemical cell 25 and chamber 22 (which is formed as a hollowed portion of base member 27) by conventional means which may include an O-ring 28 held in a sealing position by a retaining member 29 and screws 30 which secure the electrochemical cell 25 to base member 27.

4 The electrochemical cell 25 may also be threadihly attached within the retaining member 29 as at 31 to facilitate its separate removal. The desired sealing compression on O-ring 28. in that embodiment. is accomplished by screwing down cell 25.

The membrane 24 is a non-conducting material which is impervious to the passage of either electrolyte in the electrochemical cell 25 or the solution in chamber 22 and one which does not chemically react with either solution. It is highly oxygen permeable. however. and. as such. does allow the passage ofoxygen from the solution in the chamber 22 into the cell 26. One material which has successfully been used for the membrane is a film of a tetrafluroethylene polymer (such as 0.5 to l mil Emflon. UTF-lO(). manufactured by Pallflex Products Corporation of Putnam. Conn.]. The electrode 26 is positioned preferrably directly opposite the point of impingement of the solution emminating from the conduit 21 to reduce the lag time in making the oxygen measurementv The combination of the relatively high velocity impingement of the solution and the electrode location results in a considerably faster response by the electrode 26.

Leads 32 convey the signal from the electrochemical cell 25 to a conventional oxygen analyzer shown in block at 33 and the processed signals are then fed to a display device at 34.

The electrochemical oxygen detection system is also a conventional system which utilizes an electrode for determining dissolved oxygen such as a model No. 39553 manufactured by Beckman Instruments. Inc. of Fullerton. Calif. The oxygen analyzer 33 and display device 34, downstream electrically from the oxygen detecting electrochemical system may also be any con ventional instrumentation connected in a well known manner. The display device 34 may be calibrated to read the percentage of glucose in the sample directly if desired.

Conduit means l2, l7 and 21 may be glass tubing or any such conduit means conventional to analytical apparatus. The only criteria being that they not affect or be affected by the solutions passed therethrough in the preferred embodiment. The conduit 21 along with drain 23 are formed by conventional hollowed fittings with the exception of those located in the block 27, discussed below. Fittings are normally made ofa relatively inert material such as teflon or nylon and the various components are held together by screwed compression fittings as at 35, for example.

The reaction column for containing the immobilized enzyme may be a conventional glass tubing column or one of compatable plastic such as an acrylic. The gel utilized for entrapping the enzyme is a specially prepared crosslinked acrylamide gel in which the cross linking agent is normally N. N-methylenebisacrylamide and in which the ratio of acrylamide to cross linking agent is extremely high. normally from about 45:1 to about 55:1. This high ratio gel yields a much improved enzyme entrapping latice in which the leaching of the glucose oxidase molecules is greatly reduced. The preparation of the gel entrapping the glucose oxidase enzyme is described more fully in the copending application of Koch et al. Ser. No. 425.043 (which is a division of Ser. No. 276,630 filed July 3l I972, now abandoned) and assigned to the same assignee as the present application.

As discussed above. the base member 27 contains hollowed chamber 22 into which the conduit 21 feeds and out of which the drain 23 drains the fluid. The downstream end of the conduit 21 is disposed such that the fluid. as indicated by the arrows in the preferred embodiment is directed in a direction normal to the membrane 24 such that it impinges directly on that membrane. The electrode 26 is disposed. in the preferred embodiment. directly opposite the membrane lined up with the impinging stream from the conduit 21.

As briefly mentioned above. this arrangement has several advantages. First. by causing the flowing stream to impinge directly on the membrane opposite the electrode 26, oxygen depletion in the solution in the immediate vicinity of the electrode 26 associated with the consumption of oxygen by the electrode which results in possible measurement error is prevented by the continual flow of the solution. Second, the constant draining of the system also ensures that the fluid immediately in front of the membrane is constantly being changed and the entire chamber 22 is constantly being washed by new fluid. Thus, the arrangement both avoids incorrect reading of the oxygen content due to oxygen consumption from the fluid by the electrode and avoids the possibility of incorrect readings being caused from residual fluid in the chamber from previous samples. The outlet hole 21a of the conduit 21 is normally made very small to permit the operation of the entire system at a lower flow rate and greatly reduce the required sample size. In this respect the configuration of the flow system in the chamber 22 is also quite important at this reduced flow rate. For an outlet hole 21a of approximately 0.18 cm. in diameter, the normal flow rate through the entire system in the preferred embodiment is approximately 1 ml per minute and the required sample size at this flow rate is approximately 50 ul per minute per sample. As explained above, this extremely low flow rate not only conserves the carrier solution and greatly reduces the size of the required sample but also allows the system to be continually operated with valve 13 open so that the chamber 22 and membrane 24 can be continuously cleansed between tests thus returning the oxygen measurement quickly to a stable baseline between the injection of consecutive samples.

The base 27 is normally made ofa block of compatable polymer material such as an acrylic plastic which can be drilled or molded to the appropriate configuration for the system. Because of the small volume of sample and carrier solution required for each test, the entire system may be conveniently located within a small cabinet or portable analytical device. Thus, the present invention presents a simple, convenient and economical analytical instrument for the analysis of glucose in biological fluids which combines simplicity of design with speed and accuracy in glucose measurement in such fluids.

In operation, the carrier solution, as mentioned above, continuously flows through the system and the biological fluid sample containing approximately 50 ii] is injected into the flowing carrier solution through the septum 16. The carrier solution is normally an 0.1 M aqueous solution of sodium acetate buffer. The combined solution then flows by gravity through the immobilized enzyme column in which the molecules of solution are free to contact the immobilized enzyme and the above-mentioned catalyzed reaction then takes place as the solution flows through the column. At the low volume rate of flow of approximately 1 ml per min- 6 utc the reaction is essentially complete at the time the solution exits from the bottom of the column through the screen or frit 20 and passes via the conduit 2| to chamber 22 where the oxygen is measured. Thus. no electrical time delays or other complicated electronic or electrical devices are needed in the instrument to measure rate of change of oxygen concentration or to allow for initial surges in the reaction as the reaction is essentially complete before any measurement is made by the oxygen electrode 26. Thus. ofcourse. is another advantage of the simplified flow through system of the present invention.

It should also be noted that inasmuch as the rate of the enzyme-catalyzed reaction involved is somewhate temperature dependent, conventional temperature control means may be required in applications where temperature variations are like.

The embodiments ofthe invention in which an exclusive property or right is claimed are defined as follows: 1. A flow through method for determining the con centration of a component of interest in a sample wherein the detection of the concentration or change therein of a gaseous indicating species following an enzyme-catalyzed reaction is indicative of the concentra tion of the component of interest. said method comprising the steps of:

combining said sample with a carrier solution; causing the combined solution to flow past a quantity of an enzyme selected to catalyze the desired reaction. wherein said enzyme is immobilized in a manner which allows free contact between the components in said combined solution and the enzyme molecules occasioning little or no loss of enzyme thereby; after said reaction is substantially complete. causing the reacted solution to impinge upon a membrane selectively permeable to said gaseous indicating species at an angle with said membrane:

monitoring the concentration of said indicating species diffusing through said membrane;

generating an output indicative of the concentration of said indicating species;

transducing said output into one indicative of the value of the concentration of the component in the sample to be determined; and

removing said solution after contacting said membrane.

2. The method of claim 1, including the step of:

flushing the entire system with an additional amount of said carrier solution thereby regenerating the original conditions prior to the introduction of a subsequent sample.

3. The method of claim 1, including the step of:

controlling the velocity in contacting said immobilized enzyme and said diffusion membrane so that the time interval between the start of the reaction and the measurement of the concentration of the indicating species is fixed.

4. The method of claim I wherein said reacted solution is caused to impinge on said membrane such that said membrane is continually cleansed by said reacted solution.

5. A flow-through chemical analysis apparatus for determining the concentration of a component of interest in a sample wherein the detection of the concentration of an indicating species following an enzyme'catalyzed reaction is indicative of the concentration of the component of interest in the sample, said apparatus com 7 prising:

a first chamber for containing an amount of an immohilized enzyme disposed therein in a manner which allows free contact between said enzyme and a solution flowing therethrough to thereby catalyze said enzyme-catalyzed reaction;

means for introducing a carrier solution into said first chamber;

means for introducing a sample into said solution.

a second chamber for receiving said solution from said first chamber:

sensor means for detecting said indicating species in said solution. said sensor means being separated from the said second chamber by a membrane per meable to said indicating species;

flow means connecting the outlet of said first chamher with said second chamber. in a manner which causes said solution to impinge on said membrane at an angle with said membrane;

means for generating an output from said sensor related to the concentration of said indicating species;

drain means for removing said solution after contacting said membrane; and

transducing means for transducing said output into one indicative of the concentration of the component in the sample sought to be determined.

6. The apparatus of claim 5 wherein said first chamber is in the form of an enclosed packed column.

7. The apparatus as claimed in claim 5 wherein said system is a gravity flow-through system. wherein said reagent-containing solution is fed to the top of said column at an adjustable rate and wherein said second chamber is disposed in a manner which allows it to drain also by gravity.

8. The apparatus as claimed in claim 5 further comprising:

means to control the rate of flow of said solution through said first and said second chambers.

9. The apparatus as claimed in claim 5 wherein said means for supplying said sample is an injection port wherein an accurately determined amount of such sample may be injected into said solution.

10. The apparatus of claim 5 wherein said sensor is an electrochemical detector and said indicating species to be detected is a dissolved gas. said detector having a sensoring electrode disposed opposite and in close proximity to said membrane.

ll. The apparatus as claimed in claim 5 wherein the outlet of flow means is disposed in a manner which causes said solution to impinge on said membrane at right angles to said membrane opposite the location of said sensor.

Claims (11)

1. A FLOW THROUGH METHOD FOR DETERMINING THE CONCENTRATION OF A COMPONENT OF INTEREST IN A SAMPLE WHEREIN THE DETECTION OF THE CONCENTRATION OR CHANGE THEREIN OF A GASEOUS INDICATING SPECIES FOLLOWING AN ENZYME-CATALYZED REACTION IS INDICATIVE OF THE CONCENTRATION OF THE COMPONENT OF INTEREST, SAID METHOD COMPRISING THE STEPS OF: COMBINING SAID SAMPLE WITH A CARRIER SOLUTION, CAUSING THE COMBINED SOLUTION TO FLOW PAST A QUANTITY OF AN ENZYME SELECTED TO CATALYZETHE DESIRED REACTION, WHEREIN SAID ENZYME IS IMMOBILIZED IN A MANNER WHICH ALLOWS FREE CONTACT BETWEEN THE COMPONENTS IN SAID COMBINED SOLUTION AND THE ENZYME MOLECULES OCCASIONING LITTLE OR NO LOSS OF ENZYME THEREBY, AFTER SAID REACTION IS SUBSTANTIALLY COMPLETE, CAUSING THE REACTED SOLUTION TO IMPINGE UPON A MEMBRANE SELECTIVELY PERMEABLE TO SAID GASEOUS INDICATING SPECIES AT AN ANGLE WITH SAID MEMBRANE, MONITORING THE CONCENTRATION OF SAID INDICATING SPECIES DIFFUSING THROUGH SAID MEMBRANE, GENERATING AN OUTPUT INDICATIVE OF THE CONCENTRATION SAID INDICATING SPECIES,
2. The method of claim 1, including the step of: flushing the entire system with an additional amount of said carrier solution thereby regenerating the original conditions prior to the introduction of a subsequent sample.
3. The method of claim 1, including the step of: controlling the velocity in contacting said immobilized enzyme and said diffusion membrane so that the time interval between the start of the reaction and the measurement of the concentration of the indicating species is fixed.
4. The method of claim 1 wherein said reacted solution is caused to impinge on said membrane such that said membrane is continually cleansed by said reacted solution.
5. A flow-through chemical analysis apparatus for determining the concentration of a component of interest in a sample wherein the detection of the concentration of an indicating species following an enzyme-catalyzed reaction is indicative of the concentration of the component of interest in the sample, said apparatus comprising: a first chamber for containing an amount of an immobilized enzyme disposed therein in a manner which allows free contact between said enzyme and a solution flowing therethrough to thereby catalyze said enzyme-catalyzed reaction; means for introducing a carrier solution into said first chamber; means for introducing a sample into said solution; a second chamber for receiving said solution from said first chamber; sensor means for detecting said indicating species in said solution, said sensor means being separated from the said second chamber by a membrane permeable to said indicating species; flow means connecting the outlet of said first chamber with said second chamber, in a manner which causes said solution to impinge on said membrane at an angle with said membrane; means for generating an output from said sensor related to the concentration of said indicating species; drain means for removing said solution after contacting said membrane; and transducing means for transducing said output into one indicative of the concentration of the component in the sample sought to be determined.
6. THE APPARATUS OF CLAIM 5 WHEREIN SAID FIRST CHAMBER IS IN THE FORM OF AN ENCLOSED PACKED COLUMN.
7. The apparatus as claimed in claim 5 wherein said system is a gravity flow-through system, wherein said reagent-containing solution is fed to the top of said column at an adjustable rate and wherein said second chamber is disposed in a manner which allows it to drain also by gravity.
8. The apparatus as claimed in claim 5 further comprising: means to control the rate of flow of said solution through said first and said second chambers.
9. The apparatus as claimed in claim 5 wherein said means for supplying said sample is an injection port wherein an accurately determined amount of such sample may be injected into said solution.
10. The apparatus of claim 5 wherein said sensor is an electrochemical detector and said indicating species to be detected is a dissolved gas, said detector having a sensoring electrode disposed opposite and in close proximity to said membrane.
11. The apparatus as claimed in claim 5 Wherein the outlet of flow means is disposed in a manner which causes said solution to impinge on said membrane at right angles to said membrane opposite the location of said sensor.
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