US3421982A - Enzymatic analysis - Google Patents
Enzymatic analysis Download PDFInfo
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- US3421982A US3421982A US455828A US3421982DA US3421982A US 3421982 A US3421982 A US 3421982A US 455828 A US455828 A US 455828A US 3421982D A US3421982D A US 3421982DA US 3421982 A US3421982 A US 3421982A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance
- G01N27/021—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance before and after chemical transformation of the material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
- C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
- C12Q1/006—Enzyme electrodes involving specific analytes or enzymes for glucose
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
Definitions
- This invention relates to a method for enzymatic analysis, and more specifically to a method for the quantitative determination of enzymatic substrates or their enzymes, generally, though not necessarily, in the area of biological substances.
- the method forming the subject of the present invention possesses notable advantages over those hitherto known in the extreme simplicity of the reagent list called for, in the accuracy thereof, and in the rapidity with which determinations are made.
- the method and apparatus of this invention have been applied with particular facility to the determination of urea in biological fluids, such as blood serum.
- biological fluids such as blood serum.
- the apparatus functions entirely adequately with whole blood samples. It may also be employed for the determination of glucose in comparable biological fluids with the same ease and accuracy as with urea, but lat the present time an additional step is called for in the preparation of the iiuid sample.
- the apparatus and method indeed, are applicable to any other analogous enzyme system wherein ya substrate has a different conductivity from the reaction products thereof, and is suit-ed to the converse determination of enzyme concentrations as well as substrate concentrations.
- the method and .apparatus of this invention are well suited to an automated analysis wherein a number of samples maybe analyzed consecutively automatically wth the' of apparatus embodying the FIG. 3 is a front elevation of an ion exchange column which is useful in certain applications of the apparatus;
- FIG. 4 is a circuit diagram of the apparatus.
- FIGS. l and 2 show a cabinet 10, housing the mechanism and circuitry of the present apparatus, having a front face 12 on which is mounted a timer dial 14 having a timer START button 15 thereon, a meter 16, a power switch 18, an indicator light 19, a stirrer switch 20, a stirrer speed control 22, an amplifier gain control 26, a balance control 28, and fa calibrate control 24.
- the left lupper corner of the cabinet is recessed as at 30 to provide a sample table 32.
- a column 34 extends upwardly from the cabinet adjacent the recess 30.
- a forwardly extending arm 36 is mounted to the column 34 and supports, at its forward end, a pair of electrodes 38 extending downwardly therefrom, and a sleeve 39 enclosing all but the lower ends of the electrodes.
- the column 34 has bayonet slots 40 engaged by appropriate pins (not shown) on the arm 36 so that the electrodes may be lowered to a position near the table 32, or supported substantially above yand out of a sample beaker 42. In the lowered position, of course, the electrodes 38 will be immersed in whatever fluid may be contained in the beaker.
- a pair of leads 44, 46 extend from the electrodes into the interior of the cabinet through the arm 36 and column 34.
- FIG. 3 illustrates an ion exchange apparatus which iS used in certain applications of the method. It consists of burette 48 having a cotton or fiber glass plug 50 therein immediately above the stopcock 52 which supports a column of ion exchange resin 54. The burette is supported' vertically for the delivery of a sample into a beaker 42.
- a source of line alternating current 60 is connected through switch 18 to the primary 64 of a transformer 66.
- the indicator light 19 is connected to the line across the primary 64.
- the secondary 68 of the transformer forms a part of the measuring circuit 69 and is center tapped at 71, the center tap being connected to ground 70 and to lead 72.
- Lead 72 provides the ground connection for the input side of an amplifier 74.
- One half 76 of the transformer is connected through, for instance, a 5,000 ohm adjustable balance potentiometer 78 to a terminal 80.
- the other half 82 of the secondary of the transformer is connected through lead 44, the electrodes 38 (regarded, with the beaker-contained sample, as a cell 83), and lead 46 also to terminal 80.
- Terminal is also connected through a variable calibration resistor 84 which may have, for instance, a value of 50,000 ohms to one of the fixed terminals 86 of a gain potentiometer 88 which also may have a value of 50,000 ohms.
- the other fixed terminal 90 of the gain potentiometer 88 is connected to lead 72.
- the movable tap 92 of the gain potentiometer is connected to the input side of the amplifier 74.
- the movable contacts of the balance resistor 78 and of the potentiometer 88 are ganged to move together in a one-to-one ratio with, however, a declutching device 91 interposed in the ganging so as to permit independent movement of the contacts.
- clutching or declutching devices are entirely conventional, and as its specific character is not pertinent to this invention, a diagrammatic representation thereof has been deemed sufficient.
- the output lof the amplifier is connected through a full wave rectifier to the ammeter 16.
- a diode 104 may be placed across the meter for the suppression of peaks.
- a control circuit 106 consisting of the leads 108 and 110 is connected to lthe source of power in parallel with the measuring circuit 69.
- a stirrer motor circuit 112 is connected between the leads 108 and 110 and includes a stirrer motor 114, a variable resistor 22 which serves as a speed control for the motor, a stirrer switch 20, and a stirrer timer controlled switch 116.
- the stirrer timer 118 which controls switch 116 is connected between lead 108 and a terminal 120 of a double throw main timer controlled switch 122.
- the main timer 14 is connected in parallel with the stirrer timer 118 between lead 108 and terminal 120.
- Stir-rer timer controlled switch 116 is normally closed against a contact 124 connected to lead 110.
- the blade 126 of main timer controlled switch 122 is likewise connected to lead 110.
- Switch 122 includes as a Contact alternate to contact 120, contact 128 which is connected to a lead 130.
- yLead 130 includes a dropping resistor 132 and thence continues into a rectifier circ-uit connected between it and lead 106 consisting of the diode 134, capacitors 136, and resistors 138 to a meter clamp 140.
- Main timer 14 is a manually started, electrically reset timer.
- Stirrer timer 118 is electrically started and reset.
- the switches 116 and 122 are illustrated in their condition before the start of a determination.
- the main power switch 18 is closed, thereby providing power to the control circuit 106.
- the stirrer switch 20 is likewise closed and the main timer 14 is started. Starting of the main timer moves yblade 126 of switch 122 against contact 120 which de-energizes the meter clamp circuit. Simultaneously it starts the stir'- rer timer 118 which in turn closes blade 116 against contact 124.
- the stirrer thus operates for a period of time determined by the setting of the stirrer timer 118-for instance, about five seconds-at which time the stirrer timer operates to open switch 116 and resets itself.
- the main timer continues to run, for instance, for one minute when its time expires, the timer is reset, and blade 126 is closed against contact 128 to energize the meter clamp 140.
- starting of the apparatus simultaneously starts the two timers, starts the stirrer motor and releases the meter clamp. After the expiration of a few seconds, the stirrer motor is shut off.
- the main timer continues to operate for the desired determination period at the termination of which it resets and operates to energize the meter clamp.
- the timers and the detailed circuitry involved in their operation are entirely conventional and well known and play no part in this invention, the above description is believed sufficient.
- the instrument is calibrated for, for instance, a urea determination as follows. l ml. of distilled water is placed in a. beaker and a stirring bar inserted; 0.05 ml. of a standard serum containing about 50 to 70 milligrams percent of urea nitrogen is added to the water, and the beaker is placed on the table 32.
- the ganged balance resistor 78 and gain potentiometer 88 are set at mid-scale. Thereafter, the 0.5 ml. of the urease reagent are introduced into the beaker, and, as immediately as possible, the button 15 is pressed to start the operating cycle of the machine and the electrodes 38 are lowered into the beaker 42. The stirring motor, of course, will stir the solution. The balance resistor 78 should then be adjusted to bring the meter on scale. Since the balance potentiometer is ganged to the gain potentiometer, balancing the bridge automatically compensates for differences in conductivity ot the solution in the beaker. The reaction will begin immediately and the conductivity of the solution will accordingly change continuously.
- the meter should be continuously balanced to zero by adjustment of the balance resistor. ⁇ ln such continuous adjustment the knob setting of the resistor should be anticipated, such that at exactly twenty seconds after the start of the timer the needle will read zero and thereafter move up scale. The lbalance resistor is thus adjusted.
- the calibration resistor 84 As the reaction proceeds, attention is now given to the calibration resistor 84. Here the end of the total time interval of sixty seconds is anticipated, and, as this termination approaches, the calibration resistor is set to anticipate on the microammeter scale the numerical value of the milligram percent urea nitrogen of the sample. At the termination of the time interval, the meter clamps and the cycle ends; the meter clamping in the described embodiment consists of moving the meter face and needle bodily against the cover glass.
- the balance potentiometer may be disengaged from the gain potentiometer and the gain potentiometer rotated so as to bring the calibrate potentiometer into usable range. rlhe controls are then reengaged and the calibration procedure repeated.
- an unknown sample may now be analyzed.
- the unknown sample will be introduced as before by diluting 0.05 ml. of the unknown in l0 ml. of water.
- the calibrate knob remains set or locked.
- the enzyme is added in the above quantity, the timer started, and the electrodes lowered.
- the balance knob is again adjusted during the initial twenty second period to arrive at a zero reading at the end of the twenty second period with the meter needle swinging to the right as conductivity increases.
- the controls are left alone, and at the termination of the one minute cycle the meter clamps, giving a direct reading of milligram percent urea nitrogen.
- the purpose underlying the integrated balance and gain controls is that the initial conductivity of the different unknown samples may differ, and as the initial conductivity increases, the sensitivity to a change in conductivity due to the ammonium carbonate formation decreases, thus requiring an increase in gain. It has been found that regardless of initial conductivity of the sample, the rate of change of conductivity is still linear within the first minute and proportionate to the urea concentration.
- the potentiometer resistance values for the gain and balance controls have been so selected that the change in gain is proportional to change in conductivity, and direct readings will result even though samples such as serums will vary in conductivity.
- the method as applied to the determination of glucose in body fluids is essentially the same except that the decomposition product of glucose-ie., gluconic acidis substantially less highly ionized than the ammonium carbonate, and therefore a direct analysis in the presence of other normally present conductive ions is apt to lead to inaccurate results. Therefore the determination of glu- 5 cose involves the use of the ion exchange column as illustrated in FIG. 3.
- the ion exchange resin employed in this invention is a strong resin containing sulphonic cation and quaternary anion exchange resins in mixed bed form such as Amberlite MB-3, a product yof Rohm & Haas, Inc.
- Weak ion exchange resins in the past have been employed in -removing ions from biological samples. They do not affect the component to be determined-4e., the glucose-but their use requires a relatively slow passage of the sample. On the other hand, strong resins have been avoided because they are likely to affect the concentration of the unknown substance.
- the treatment time has been greatly shortened, and it has been found that the strong resin can be adjusted in quantity and a grain size chosen to determine the period of exposure of the sample to the resin so that the interfering ions are removed, but the glucose content is not disturbed.
- the ion exchange column is prepared by depositing the cotton or glass plug 50 in the bottom thereof and there- .after pouring in a suspension of the resin 54 in water. The water is drained off to a point where the level thereof is slightly above the settled resin bed. The bed should never -be permitted to dry.
- the enzyme solution is prepared by dissolving one gram of glucose oxidase of 15,000 activity units per gram in 25 m1. of water. The solution is passed through an ion exchange column prepared as described above, centrifuged, and diluted with an equal volume of glycerol.
- 0.1 cc. of the sample i.e., blood, serum, delibrinated blood, etc.-is added to the water standing above the resin in the ion exchange column (not the same column as used for the enzyme filtration).
- the sample is carried through the column by the slow addition of 50 ml. water.
- the initial introduction of the water should be particularly careful and gradual in order that the sample be carried through and not be diluted by the whole 50 ml. quantity.
- l ml. of the diluted sample as iltered through the ion exchange column is introduced into the sample beaker and 0.5 cc. of the enzyme solution is added to it. Thereafter the procedure is followed as with the urea determination, following the general sample preparation as described immediately above and the actual balancing of the apparatus as described in conjunction with the urea determination.
- the apparatus will be differently and Iindependently calibrated for glucose determinations, of course.
- the advantages of this method and apparatus will be readily apparent from the foregoing description.
- the method is notable for its quickness and accuracy of determination in that the sample preparation is exceedingly quick, the reagent list small, and the actual analysis period only one minute.
- the method requires the use of an exceedingly small sample, 0.05 to 0.1 cc. being all that is required.
- Urea determinations have been successfully performed with blood samples as small as 0.02 cc. It is, of course, by virtue of the fact that conductivity is measured that the necessity for any reagents other than the enzyme is eliminated. Also, by determining the rate of reaction, the determination can be made in the aforesaid one minute period even though the reaction itself continues for a substantially longer period of time.
- the device of this invention is well suited to autotmatic operation.
- a machine has been devised wherein a large number of beakers containing biological samples to be analyzed are carried on a rotating table such that each of the beakers passes into position under the electrodes in successive order. As the table is indexed around to position each beaker, the electrodes are lowered into the consecutive beakers and removed therefrom, rinsed olf, and lowered again into the next beaker.
- the readout may be printed on a tape so as to avoid any need for constant supervision of the process.
- the invention here resides primarily in the method and in those aspects of the apparatus relating to the method, such automatic machine has not been illustrated.
- An analytical method for the quantitative determination of either of the elements in an enzyme-substrate system wherein the reaction product has a conductivity different from that of the substrate which comprises mixing only solutions of the enzyme and substrate, one of which is a quantitative known, immersing a pair of electrodes having a voltage thereacross in the mixture and reading the variation in current iiow between said electrodes in a predetermined period of time within that period during which the rate of reaction is linear.
- An analytical method for the quantitative determination of either of the elements in an enzyme-substrate system wherein the reaction product has a conductivity different from that of the substrate which comprises mixing only solutions of the enzyme and substrate, one of which is a quantitative known, and immersing a pair of electrodes having a voltage thereacross constituting one leg of a bridge circiuit in the mixture, balancing the bridge reading to zero, reading the degree of unbalance of the bridge a predetermined time after the balancing thereof, said balancing and said reading of the unbalance both being performed within that period during which the rate of reaction is linear.
- a method for the determination of urea in a biological uid which comprises mixing only a solution of the fluid with a solution of a known quantity of urease enzyme, immersing a pair of electrodes having a voltage thereacross in the mixture and reading the variation in current ow between said electrodes in a predetermined period of time within that period during which the rate of reaction is linear.
- a method for determining glucose in a biological fluid which comprises making an aqueous solution of said fluid, passing said solution quickly through a strong mixed bed ion exchange resin to remove unwanted ions from said solution, mixing said solution with a solution having a known quantity of glucose oxidase therein, immersing a pair of electrodes having a voltage thereacross in the mixture of solutions and reading the variation in current flow between said electrodes in a predetermined period of time within that period during which the rate of reaction is linear.
- a method for the quantitative determination of either of the elements in a urea-urease system which comprises mixing only a solution of the urea with a solution of the urease, one of which is a quantitative known, immersing a pair of electrodes having a voltage thereacross in the mixture and reading the variation in ,current ow between said electrodes in a predetermined period of time within that period during which the rate of reaction is linear.
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Description
Jan. 14, 1969 F. C. SCHULTZ ET AL ENZYMATIC ANALYSIS Filed May 14, 1965 @faz/enfans @w72 PMM United States Patent 3,421,982 ENZYMATIC ANALYSIS Frederick C. Schultz and Gordon P. McFaul, St. Joseph, Mich., and Franklin Lim, Richmond, Va., assignors to Laboratory Equipment Corporation, St. Joseph, Mich., a corporation of Michigan Filed May 14, 1965, Ser. No. 455,828 U.S. Cl. 195-103.5 Int. Cl. G01n 27 00 8 Claims ABSTRACT F THE DISCLOSURE This invention relates to a method for enzymatic analysis, and more specifically to a method for the quantitative determination of enzymatic substrates or their enzymes, generally, though not necessarily, in the area of biological substances.
The method forming the subject of the present invention possesses notable advantages over those hitherto known in the extreme simplicity of the reagent list called for, in the accuracy thereof, and in the rapidity with which determinations are made.
The method and apparatus of this invention have been applied with particular facility to the determination of urea in biological fluids, such as blood serum. As a matter of fact, the apparatus functions entirely adequately with whole blood samples. It may also be employed for the determination of glucose in comparable biological fluids with the same ease and accuracy as with urea, but lat the present time an additional step is called for in the preparation of the iiuid sample. The apparatus and method, indeed, are applicable to any other analogous enzyme system wherein ya substrate has a different conductivity from the reaction products thereof, and is suit-ed to the converse determination of enzyme concentrations as well as substrate concentrations.
The method and .apparatus of this invention are well suited to an automated analysis wherein a number of samples maybe analyzed consecutively automatically wth the' of apparatus embodying the FIG. 3 is a front elevation of an ion exchange column which is useful in certain applications of the apparatus; and
FIG. 4 is a circuit diagram of the apparatus.
FIGS. l and 2 show a cabinet 10, housing the mechanism and circuitry of the present apparatus, having a front face 12 on which is mounted a timer dial 14 having a timer START button 15 thereon, a meter 16, a power switch 18, an indicator light 19, a stirrer switch 20, a stirrer speed control 22, an amplifier gain control 26, a balance control 28, and fa calibrate control 24.
The left lupper corner of the cabinet is recessed as at 30 to provide a sample table 32. A column 34 extends upwardly from the cabinet adjacent the recess 30. A forwardly extending arm 36 is mounted to the column 34 and supports, at its forward end, a pair of electrodes 38 extending downwardly therefrom, and a sleeve 39 enclosing all but the lower ends of the electrodes. The column 34 has bayonet slots 40 engaged by appropriate pins (not shown) on the arm 36 so that the electrodes may be lowered to a position near the table 32, or supported substantially above yand out of a sample beaker 42. In the lowered position, of course, the electrodes 38 will be immersed in whatever fluid may be contained in the beaker. A pair of leads 44, 46 extend from the electrodes into the interior of the cabinet through the arm 36 and column 34.
FIG. 3 illustrates an ion exchange apparatus which iS used in certain applications of the method. It consists of burette 48 having a cotton or fiber glass plug 50 therein immediately above the stopcock 52 which supports a column of ion exchange resin 54. The burette is supported' vertically for the delivery of a sample into a beaker 42.
The wiring diagram is illustrated in FIG. 4. A source of line alternating current 60 is connected through switch 18 to the primary 64 of a transformer 66. The indicator light 19 is connected to the line across the primary 64. The secondary 68 of the transformer forms a part of the measuring circuit 69 and is center tapped at 71, the center tap being connected to ground 70 and to lead 72. Lead 72 provides the ground connection for the input side of an amplifier 74. One half 76 of the transformer is connected through, for instance, a 5,000 ohm adjustable balance potentiometer 78 to a terminal 80. The other half 82 of the secondary of the transformer is connected through lead 44, the electrodes 38 (regarded, with the beaker-contained sample, as a cell 83), and lead 46 also to terminal 80.
Terminal is also connected through a variable calibration resistor 84 which may have, for instance, a value of 50,000 ohms to one of the fixed terminals 86 of a gain potentiometer 88 which also may have a value of 50,000 ohms. The other fixed terminal 90 of the gain potentiometer 88 is connected to lead 72. The movable tap 92 of the gain potentiometer is connected to the input side of the amplifier 74.
The movable contacts of the balance resistor 78 and of the potentiometer 88 are ganged to move together in a one-to-one ratio with, however, a declutching device 91 interposed in the ganging so as to permit independent movement of the contacts. As such clutching or declutching devices are entirely conventional, and as its specific character is not pertinent to this invention, a diagrammatic representation thereof has been deemed sufficient.
The output lof the amplifier is connected through a full wave rectifier to the ammeter 16. A diode 104 may be placed across the meter for the suppression of peaks.
A control circuit 106 consisting of the leads 108 and 110 is connected to lthe source of power in parallel with the measuring circuit 69. A stirrer motor circuit 112 is connected between the leads 108 and 110 and includes a stirrer motor 114, a variable resistor 22 which serves as a speed control for the motor, a stirrer switch 20, and a stirrer timer controlled switch 116. The stirrer timer 118 which controls switch 116 is connected between lead 108 and a terminal 120 of a double throw main timer controlled switch 122. The main timer 14 is connected in parallel with the stirrer timer 118 between lead 108 and terminal 120. Stir-rer timer controlled switch 116 is normally closed against a contact 124 connected to lead 110. The blade 126 of main timer controlled switch 122 is likewise connected to lead 110. Switch 122 includes as a Contact alternate to contact 120, contact 128 which is connected to a lead 130. yLead 130 includes a dropping resistor 132 and thence continues into a rectifier circ-uit connected between it and lead 106 consisting of the diode 134, capacitors 136, and resistors 138 to a meter clamp 140.
Main timer 14 is a manually started, electrically reset timer. Stirrer timer 118 is electrically started and reset. The switches 116 and 122 are illustrated in their condition before the start of a determination. To initiate a cycle or a determination, the main power switch 18 is closed, thereby providing power to the control circuit 106. The stirrer switch 20 is likewise closed and the main timer 14 is started. Starting of the main timer moves yblade 126 of switch 122 against contact 120 which de-energizes the meter clamp circuit. Simultaneously it starts the stir'- rer timer 118 which in turn closes blade 116 against contact 124. The stirrer thus operates for a period of time determined by the setting of the stirrer timer 118-for instance, about five seconds-at which time the stirrer timer operates to open switch 116 and resets itself. The main timer continues to run, for instance, for one minute when its time expires, the timer is reset, and blade 126 is closed against contact 128 to energize the meter clamp 140. In short, starting of the apparatus simultaneously starts the two timers, starts the stirrer motor and releases the meter clamp. After the expiration of a few seconds, the stirrer motor is shut off. The main timer continues to operate for the desired determination period at the termination of which it resets and operates to energize the meter clamp. As the timers and the detailed circuitry involved in their operation are entirely conventional and well known and play no part in this invention, the above description is believed sufficient.
The most significant distinction of this invention over enzymatic analytical procedures hitherto followed is that the method is based on the rate of increase of electrical conductivity attendant upon the enzymatic action. Urease converts urea to ammonium carbonate, and glucose oxidase converts glucose to gluconic acid. Although the conversion of the substrate proceeds for several minutes, the rate of change of the conductivity in the first minute is linear, the rate varying with the concentration of the enzyme and the substrate. 'It is during the first minute that the rate of change is measured in this apparatus and procedure.
The instrument is calibrated for, for instance, a urea determination as follows. l ml. of distilled water is placed in a. beaker and a stirring bar inserted; 0.05 ml. of a standard serum containing about 50 to 70 milligrams percent of urea nitrogen is added to the water, and the beaker is placed on the table 32.
The ganged balance resistor 78 and gain potentiometer 88 are set at mid-scale. Thereafter, the 0.5 ml. of the urease reagent are introduced into the beaker, and, as immediately as possible, the button 15 is pressed to start the operating cycle of the machine and the electrodes 38 are lowered into the beaker 42. The stirring motor, of course, will stir the solution. The balance resistor 78 should then be adjusted to bring the meter on scale. Since the balance potentiometer is ganged to the gain potentiometer, balancing the bridge automatically compensates for differences in conductivity ot the solution in the beaker. The reaction will begin immediately and the conductivity of the solution will accordingly change continuously. The meter should be continuously balanced to zero by adjustment of the balance resistor. `ln such continuous adjustment the knob setting of the resistor should be anticipated, such that at exactly twenty seconds after the start of the timer the needle will read zero and thereafter move up scale. The lbalance resistor is thus adjusted. As the reaction proceeds, attention is now given to the calibration resistor 84. Here the end of the total time interval of sixty seconds is anticipated, and, as this termination approaches, the calibration resistor is set to anticipate on the microammeter scale the numerical value of the milligram percent urea nitrogen of the sample. At the termination of the time interval, the meter clamps and the cycle ends; the meter clamping in the described embodiment consists of moving the meter face and needle bodily against the cover glass.
In the event the calibrate knob cannot be turned far enough in either direction to yield direct readings on the microammeter, the balance potentiometer may be disengaged from the gain potentiometer and the gain potentiometer rotated so as to bring the calibrate potentiometer into usable range. rlhe controls are then reengaged and the calibration procedure repeated.
The apparatus lbeing thus balanced, an unknown sample may now be analyzed. The unknown sample will be introduced as before by diluting 0.05 ml. of the unknown in l0 ml. of water. The calibrate knob remains set or locked. The enzyme is added in the above quantity, the timer started, and the electrodes lowered. As the reaction proceeds, the balance knob is again adjusted during the initial twenty second period to arrive at a zero reading at the end of the twenty second period with the meter needle swinging to the right as conductivity increases. After the twenty second period, the controls are left alone, and at the termination of the one minute cycle the meter clamps, giving a direct reading of milligram percent urea nitrogen.
The purpose underlying the integrated balance and gain controls is that the initial conductivity of the different unknown samples may differ, and as the initial conductivity increases, the sensitivity to a change in conductivity due to the ammonium carbonate formation decreases, thus requiring an increase in gain. It has been found that regardless of initial conductivity of the sample, the rate of change of conductivity is still linear within the first minute and proportionate to the urea concentration. The potentiometer resistance values for the gain and balance controls have been so selected that the change in gain is proportional to change in conductivity, and direct readings will result even though samples such as serums will vary in conductivity.
The above application of this apparatus and method is directed specifically to the determination of urea. This particular instance was chosen because it illustrates the method in its simplest form and in what is expected will be one of its most frequent applications. The increasing proportion of ammonium carbonate, a good conductor, gives clear-cut results against a background of normal random conductive elements likely to be found in blood samples.
It will be appreciated that by the use of standard sera, the enzymatic activity, and therefore the urease content of any unknown, may be as easily determined.
The method as applied to the determination of glucose in body fluids is essentially the same except that the decomposition product of glucose-ie., gluconic acidis substantially less highly ionized than the ammonium carbonate, and therefore a direct analysis in the presence of other normally present conductive ions is apt to lead to inaccurate results. Therefore the determination of glu- 5 cose involves the use of the ion exchange column as illustrated in FIG. 3.
The ion exchange resin employed in this invention is a strong resin containing sulphonic cation and quaternary anion exchange resins in mixed bed form such as Amberlite MB-3, a product yof Rohm & Haas, Inc. Weak ion exchange resins in the past have been employed in -removing ions from biological samples. They do not affect the component to be determined-4e., the glucose-but their use requires a relatively slow passage of the sample. On the other hand, strong resins have been avoided because they are likely to affect the concentration of the unknown substance. By the use of a relatively small quantity of a strong resin, the treatment time has been greatly shortened, and it has been found that the strong resin can be adjusted in quantity and a grain size chosen to determine the period of exposure of the sample to the resin so that the interfering ions are removed, but the glucose content is not disturbed.
The ion exchange column is prepared by depositing the cotton or glass plug 50 in the bottom thereof and there- .after pouring in a suspension of the resin 54 in water. The water is drained off to a point where the level thereof is slightly above the settled resin bed. The bed should never -be permitted to dry.
The enzyme solution is prepared by dissolving one gram of glucose oxidase of 15,000 activity units per gram in 25 m1. of water. The solution is passed through an ion exchange column prepared as described above, centrifuged, and diluted with an equal volume of glycerol.
For the glucose determination, 0.1 cc. of the samplei.e., blood, serum, delibrinated blood, etc.-is added to the water standing above the resin in the ion exchange column (not the same column as used for the enzyme filtration). The sample is carried through the column by the slow addition of 50 ml. water. The initial introduction of the water should be particularly careful and gradual in order that the sample be carried through and not be diluted by the whole 50 ml. quantity.
For the analysis, l ml. of the diluted sample as iltered through the ion exchange column is introduced into the sample beaker and 0.5 cc. of the enzyme solution is added to it. Thereafter the procedure is followed as with the urea determination, following the general sample preparation as described immediately above and the actual balancing of the apparatus as described in conjunction with the urea determination. The apparatus will be differently and Iindependently calibrated for glucose determinations, of course.
The advantages of this method and apparatus will be readily apparent from the foregoing description. The method is notable for its quickness and accuracy of determination in that the sample preparation is exceedingly quick, the reagent list small, and the actual analysis period only one minute. The method requires the use of an exceedingly small sample, 0.05 to 0.1 cc. being all that is required. Urea determinations have been successfully performed with blood samples as small as 0.02 cc. It is, of course, by virtue of the fact that conductivity is measured that the necessity for any reagents other than the enzyme is eliminated. Also, by determining the rate of reaction, the determination can be made in the aforesaid one minute period even though the reaction itself continues for a substantially longer period of time.
Particular attention is also called to the use of the strong ion exchange resin in conjunction with the glucose determination whereby the quantity of resin required is small and the time of sample treatment is greatly accelerated without impairing or affecting the glucose propor- 4tion in the sample.
The device of this invention is well suited to autotmatic operation. A machine has been devised wherein a large number of beakers containing biological samples to be analyzed are carried on a rotating table such that each of the beakers passes into position under the electrodes in successive order. As the table is indexed around to position each beaker, the electrodes are lowered into the consecutive beakers and removed therefrom, rinsed olf, and lowered again into the next beaker. The readout may be printed on a tape so as to avoid any need for constant supervision of the process. However, since it is believed that the invention here resides primarily in the method and in those aspects of the apparatus relating to the method, such automatic machine has not been illustrated.
It will be appreciated that an embodiment only of the method and device has been described here, and that alternatives as to materials treated, the steps of the method, and the structural details of the device will suggest themselves. This invention therefore should be regarded as being limited only as set forth in the following claims.
We claim:
1. An analytical method for the quantitative determination of either of the elements in an enzyme-substrate system wherein the reaction product has a conductivity different from that of the substrate, which comprises mixing only solutions of the enzyme and substrate, one of which is a quantitative known, immersing a pair of electrodes having a voltage thereacross in the mixture and reading the variation in current iiow between said electrodes in a predetermined period of time within that period during which the rate of reaction is linear.
2. An analytical method for the quantitative determination of either of the elements in an enzyme-substrate system wherein the reaction product has a conductivity different from that of the substrate, which comprises mixing only solutions of the enzyme and substrate, one of which is a quantitative known, and immersing a pair of electrodes having a voltage thereacross constituting one leg of a bridge circiuit in the mixture, balancing the bridge reading to zero, reading the degree of unbalance of the bridge a predetermined time after the balancing thereof, said balancing and said reading of the unbalance both being performed within that period during which the rate of reaction is linear.
3. The method as set forth in claim 2 including the preliminary step of Calibrating the -degree of bridge unbalance against standard samples to cause bridge unbalance to read directly in parts of the unknown.
4. A method for the determination of urea in a biological uid which comprises mixing only a solution of the fluid with a solution of a known quantity of urease enzyme, immersing a pair of electrodes having a voltage thereacross in the mixture and reading the variation in current ow between said electrodes in a predetermined period of time within that period during which the rate of reaction is linear.
S. The method as set forth in claim 4 wherein the reading of current iiow variation is made no longer than about one minute from the time of the mixing of the fluid and enzyme solution.
6. A method for determining glucose in a biological fluid which comprises making an aqueous solution of said fluid, passing said solution quickly through a strong mixed bed ion exchange resin to remove unwanted ions from said solution, mixing said solution with a solution having a known quantity of glucose oxidase therein, immersing a pair of electrodes having a voltage thereacross in the mixture of solutions and reading the variation in current flow between said electrodes in a predetermined period of time within that period during which the rate of reaction is linear.
7. A method for the quantitative determination of either of the elements in a urea-urease system which comprises mixing only a solution of the urea with a solution of the urease, one of which is a quantitative known, immersing a pair of electrodes having a voltage thereacross in the mixture and reading the variation in ,current ow between said electrodes in a predetermined period of time within that period during which the rate of reaction is linear.
7 8 8. A method for the quantitative determination of References Cited either of the elementsof a glucose-glucose oxidase system FOREIGN PATENTS which comprlses m1x1ng only an lon-free solution of the glucose with a solution of the glucose-oxidase, one of 231,262 11/1960 Austral@ which solutions is a quantitative known, immersing a pair of electrodes having 'a voltage thereacross in said mixture and reading the variation in current ow between said U s C1 X R electrode in a predetermined period of time within that period during which the rate of reaction is linear. 23--25 3; 195-127; 204-195 5 ALVIN E. TANENHOLTZ, Primary Examiner.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US45582865A | 1965-05-14 | 1965-05-14 |
Publications (1)
Publication Number | Publication Date |
---|---|
US3421982A true US3421982A (en) | 1969-01-14 |
Family
ID=23810434
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US455828A Expired - Lifetime US3421982A (en) | 1965-05-14 | 1965-05-14 | Enzymatic analysis |
Country Status (2)
Country | Link |
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US (1) | US3421982A (en) |
GB (1) | GB1073767A (en) |
Cited By (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3506405A (en) * | 1965-08-27 | 1970-04-14 | Precision Scient Co | Automatic titration instrument |
US3605010A (en) * | 1969-07-14 | 1971-09-14 | Becton Dickinson Co | Automatic lysis detection apparatus |
US3746984A (en) * | 1970-09-08 | 1973-07-17 | Triplett Corp | Autopolarity voltmeter circuit |
US3857771A (en) * | 1967-02-27 | 1974-12-31 | Beckman Instruments Inc | Rate sensing batch analyzer |
US3933593A (en) * | 1971-02-22 | 1976-01-20 | Beckman Instruments, Inc. | Rate sensing batch analysis method |
US3957592A (en) * | 1974-07-29 | 1976-05-18 | Owens-Illinois, Inc. | Measurement of polarographic current |
US4021199A (en) * | 1974-07-04 | 1977-05-03 | Mitsui Mining & Smelting Co., Ltd. | Automatic cleaning apparatus for electrodes of pH meter |
US4045296A (en) * | 1972-08-09 | 1977-08-30 | Beckman Instruments, Inc. | Rate sensing batch analysis method and enzyme used therein |
US4204037A (en) * | 1978-04-04 | 1980-05-20 | Nasa | Method and automated apparatus for detecting coliform organisms |
US4260680A (en) * | 1977-10-22 | 1981-04-07 | Mitsubishi Chemical Industries, Ltd. | Method and apparatus for the measurement of glucose content |
US4271001A (en) * | 1978-03-23 | 1981-06-02 | Asahi Kasei Kogyo Kabushiki Kaisha | Apparatus for measuring membrane characteristics of vesicles |
US4314029A (en) * | 1979-02-07 | 1982-02-02 | Ohtake Works Company, Ltd. | Apparatus for automatically measuring changes in amount of gas |
FR2489520A1 (en) * | 1980-09-04 | 1982-03-05 | Applied Med Tech Inc | EXAMINING APPARATUS AND METHOD FOR MEASURING IONS IN FLUIDS |
US4560534A (en) * | 1983-11-02 | 1985-12-24 | Miles Laboratories, Inc. | Polymer catalyst transducers |
US4642559A (en) * | 1983-12-12 | 1987-02-10 | Texaco, Inc. | Electrostatic field meter |
US4672322A (en) * | 1984-08-24 | 1987-06-09 | Milton Roy Company | High sensitivity conductivity detector |
US5120648A (en) * | 1988-05-26 | 1992-06-09 | Lim Technology Laboratories, Inc. | Chemical analyzer using rf radiation attenuation measurements |
-
1965
- 1965-05-14 US US455828A patent/US3421982A/en not_active Expired - Lifetime
-
1966
- 1966-05-13 GB GB21475/66A patent/GB1073767A/en not_active Expired
Cited By (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3506405A (en) * | 1965-08-27 | 1970-04-14 | Precision Scient Co | Automatic titration instrument |
US3857771A (en) * | 1967-02-27 | 1974-12-31 | Beckman Instruments Inc | Rate sensing batch analyzer |
US3605010A (en) * | 1969-07-14 | 1971-09-14 | Becton Dickinson Co | Automatic lysis detection apparatus |
US3746984A (en) * | 1970-09-08 | 1973-07-17 | Triplett Corp | Autopolarity voltmeter circuit |
US3933593A (en) * | 1971-02-22 | 1976-01-20 | Beckman Instruments, Inc. | Rate sensing batch analysis method |
US4045296A (en) * | 1972-08-09 | 1977-08-30 | Beckman Instruments, Inc. | Rate sensing batch analysis method and enzyme used therein |
US4021199A (en) * | 1974-07-04 | 1977-05-03 | Mitsui Mining & Smelting Co., Ltd. | Automatic cleaning apparatus for electrodes of pH meter |
US3957592A (en) * | 1974-07-29 | 1976-05-18 | Owens-Illinois, Inc. | Measurement of polarographic current |
US4260680A (en) * | 1977-10-22 | 1981-04-07 | Mitsubishi Chemical Industries, Ltd. | Method and apparatus for the measurement of glucose content |
US4271001A (en) * | 1978-03-23 | 1981-06-02 | Asahi Kasei Kogyo Kabushiki Kaisha | Apparatus for measuring membrane characteristics of vesicles |
US4204037A (en) * | 1978-04-04 | 1980-05-20 | Nasa | Method and automated apparatus for detecting coliform organisms |
US4314029A (en) * | 1979-02-07 | 1982-02-02 | Ohtake Works Company, Ltd. | Apparatus for automatically measuring changes in amount of gas |
FR2489520A1 (en) * | 1980-09-04 | 1982-03-05 | Applied Med Tech Inc | EXAMINING APPARATUS AND METHOD FOR MEASURING IONS IN FLUIDS |
US4560534A (en) * | 1983-11-02 | 1985-12-24 | Miles Laboratories, Inc. | Polymer catalyst transducers |
US4642559A (en) * | 1983-12-12 | 1987-02-10 | Texaco, Inc. | Electrostatic field meter |
US4672322A (en) * | 1984-08-24 | 1987-06-09 | Milton Roy Company | High sensitivity conductivity detector |
US5120648A (en) * | 1988-05-26 | 1992-06-09 | Lim Technology Laboratories, Inc. | Chemical analyzer using rf radiation attenuation measurements |
Also Published As
Publication number | Publication date |
---|---|
GB1073767A (en) | 1967-06-28 |
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