U.S. patent number 3,765,841 [Application Number 05/169,687] was granted by the patent office on 1973-10-16 for method and apparatus for chemical analysis.
This patent grant is currently assigned to Beckman Instruments, Inc.. Invention is credited to Gerald L. Paulson, Robert A. Ray.
United States Patent |
3,765,841 |
Paulson , et al. |
October 16, 1973 |
METHOD AND APPARATUS FOR CHEMICAL ANALYSIS
Abstract
A method and apparatus for determining the concentration of a
component in a sample, i.e. the concentration of urea in biological
fluids, such as blood serum, wherein the sample, upon being
introduced into solution with a reagent, reacts therewith, causing
a continuing change in a characteristic of the solution, and
wherein the rate of the reaction is indicative of the concentration
of the component in the sample. A sensor is provided for monitoring
the characteristic of the solution and for generating a first
electrical output signal proportional thereto. Differentiator
circuit means are provided for producing a second electrical signal
proportional to the time derivative of the first signal, the time
derivative signal being indicative of the concentration of the
component in the sample.
Inventors: |
Paulson; Gerald L. (Anaheim,
CA), Ray; Robert A. (Fullerton, CA) |
Assignee: |
Beckman Instruments, Inc.
(Fullerton, CA)
|
Family
ID: |
22616754 |
Appl.
No.: |
05/169,687 |
Filed: |
August 6, 1971 |
Current U.S.
Class: |
205/777.5;
422/82.02; 422/82.09; 436/108; 435/287.1; 422/82.04; 435/14;
324/442; 205/792; 204/403.01; 204/403.14 |
Current CPC
Class: |
G01N
27/3271 (20130101); Y10T 436/171538 (20150115) |
Current International
Class: |
G01N
33/487 (20060101); C12k 001/10 (); G01n 027/06 ();
G01n 033/16 () |
Field of
Search: |
;23/23R,253R
;195/103.5,127 ;324/3B,30 ;204/195 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Reese; Robert M.
Claims
We claim:
1. A method for determining the concentration of a component in a
sample, wherein the sample, upon being introduced into solution
with a reagent, reacts therewith, the rate of the reaction being
indicative of said concentration, comprising:
monitoring a characteristic of said solution or a component or
product of said reaction which is proportional to said
concentration;
generating an output signal proportional to the time rate of change
of said characteristic;
measuring the value of said output signal; and
inhibiting the measurement of the value of said output signal for a
predetermined, fixed time interval from introduction of said sample
into said reagent, said time interval being sufficient to permit
thorough mixing of said sample with said reagent.
2. A method according to claim 1 wherein said fixed time interval
is long enough to permit thorough mixing of said sample with said
reagent.
3. In a chemical analyzing system for determining the concentration
of a component in a sample, wherein said sample, upon being
introduced into solution with a reagent, reacts therewith, the rate
of the reaction being indicative of said concentration, such system
comprising sensor means for monitoring a characteristic of said
solution or a component or a product of said reaction and for
producing a first electrical output signal proportional thereto,
and differentiator circuit means for producing a second electrical
signal proportional to the time derivative of said first signal,
said time derivative signal being indicative of said concentration
of said component in said sample, the improvement comprising:
means for inhibiting operation of said differentiator circuit means
for a predetermined, fixed time interval from introduction of said
sample into said reagent, said time interval being sufficient to
permit thorough mixing of said sample with said reagent.
4. In a chemical analyzing system for determining the concentration
of a component in a sample, wherein said sample, upon being
introduced into solution with a reagent, reacts therewith, the rate
of the reaction being indicative of said concentration, such system
comprising sensor means for monitoring a characteristic of said
solution or a component or a product of said reaction and for
producing a first electrical output signal proportional thereto,
differentiator circuit means for producing a second electrical
signal proportional to the time derivative of said first signal,
said time derivative signal being indicative of said concentration
of said component in said sample, and means responsive to said
second signal for generating an output signal indicative of the
value thereof, the improvement comprising:
means for inhibiting the generation of said output signal for a
predetermined, fixed time interval initiated automatically upon
introduction of said sample into said reagent, said time interval
being sufficient to eliminate the effect of transients occurring
upon introduction of said sample into said reagent.
5. In a chemical analyzing system according to claim 4, the
improvement wherein said inhibiting means inhibits the operation of
said differentiator circuit means during said fixed time
interval.
6. In a chemical analyzing system according to claim 4, the
improvement wherein said inhibiting means inhibits the operation of
said generating means during said fixed time interval.
7. In a chemical analyzing system for determining the concentration
of a component in a sample, wherein said sample, upon being
introduced into solution with a reagent, reacts therewith, the rate
of the reaction being indicative of said concentration, such system
comprising sensor means for monitoring a characteristic of said
solution or a component or a product of said reaction and for
producing a first electrical output signal proportional thereto,
differentiator circuit means for producing a second electrical
signal proportional to the time derivative of said first signal,
said time derivative signal being indicative of said concentration
of said component in said sample, and means for measuring the value
of said time derivative signal, the improvement comprising:
means for inhibiting the measurement of said time derivative signal
for a predetermined, fixed time interval from introduction of said
sample into said reagent, said time interval being sufficient to
eliminate the effect of transients occurring upon introduction of
said sample into said reagent.
8. In a chemical analyzing system according to claim 7, the
improvement wherein said fixed time interval is long enough to
permit thorough mixing of said sample with said reagent.
9. In a chemical analyzing system according to claim 7, the
improvement wherein the rate of said reaction is not necessarily
linear.
10. In a chemical analyzing system according to claim 7, the
improvement wherein said inhibiting means inhibits the operation of
said differentiator circuit means during said fixed time
interval.
11. In a chemical analyzing system according to claim 7, the
improvement wherein said inhibiting means inhibits the operation of
said measuring means during said fixed time interval.
12. A chemical analyzer comprising:
means for receiving a sample and a reagent;
sensor means operatively associated with said receiving means for
monitoring the concentration of a component or product of the
reaction between said sample and said reagent and for producing a
first output signal proportional to said concentration;
differentiator circuit means coupled to said sensor means and
responsive to said first output signal for producing a second
output signal proportional to the time derivative of said first
output signal and thus proportional to the time rate of change of
concentration of said component or product;
means coupled to said differentiator circuit means for measuring
the value of said second signal; and
means coupled to said sensor means for inhibiting the measurement
of the value of said second signal for a predetermined, fixed time
interval from introduction of said sample and said reagent into
said receiving means, said time interval being sufficient to
eliminate the effect of transients occurring upon introduction of
said sample and said reagent into said receiving means.
13. A chemical analyzer according to claim 12 wherein said
inhibiting means comprises:
timing means responsive to an abrupt change in said first output
signal for producing a control signal, a characteristic of which
changes after said predetermined, fixed time interval; and wherein
said measuring means comprises:
means responsive to said second output signal and operative upon
the occurrence of said change in said characteristic of said
control signal for determining the value of said second output
signal.
14. A chemical analyzer according to claim 13 wherein said control
signal is applied to said differentiator circuit means for
inhibiting the operation thereof during said fixed time interval
and wherein said measuring means measures the maximum value of said
second output signal.
15. A chemical analyzer according to claim 13 wherein said timing
means produces a second control signal, a characteristic of which
changes before the termination of said fixed time interval, wherein
said second control signal is applied to said differentiator
circuit means for inhibiting the operation thereof during a first
portion of said fixed time interval, and wherein said
first-mentioned control signal is applied to said measuring means
for inhibiting the operation thereof during said fixed time
interval.
16. A chemical analyzer according to claim 12 wherein said sensor
means comprises:
first and second electrodes extending into said receiving means for
monitoring the conductance of the solution therein, wherein:
said second output signal is proportional to the rate of change of
conductance of said solution, and wherein:
said measuring means measures the value of said rate of change of
conductance after said predetermined, fixed time interval.
17. A chemical analyzer according to claim 16 further
comprising:
oscillator means operatively coupled to one of said electrodes of
said sensor means, said oscillator means generating an AC output
signal; and
demodulator means operatively coupled to the other of said
electrodes of said sensor means, said demodulator means receiving
an amplitude modulated signal and producing a DC signal which
comprises said second output signal.
18. A chemical analyzer according to claim 16 wherein said sensor
means includes a surface which is exposed to said solution, and
wherein said electrodes comprise first and second conductive areas
positioned on said surface, said conductive areas being spaced
apart.
19. A chemical analyzer according to claim 16 wherein said sensor
means includes a surface which is exposed to said solution, said
surface conforming to a segment of a sphere, and wherein said
electrodes comprise first and second conductive areas positioned on
said surface.
20. A chemical analyzer according to claim 19 wherein said
conductive areas have the shape of half circles and are positioned
with their straight sides parallel and spaced apart.
21. In a chemical analyzing system for determing the concentration
of a component in a sample, wherein said sample, upon being
introduced into solution with a reagent, reacts therewith causing
an instantaneous change in a characteristic of said solution or a
component or a product of said reaction, the rate of the reaction
being indicative of said concentration, such system comprising
sensor means for monitoring said characteristic, component or
product and for producing a first electrical output signal
proportional thereto, said change being sensed by said sensor means
producing an abrupt change in said first output signal, and
differentiator circuit means for producing a second electrical
signal proportional to the time derivative of said first signal,
said time derivative signal being indicative of said concentration
of said component in said sample, the improvement comprising:
timing means responsive to an abrupt change in said first signal
for producing a control signal, a characteristic of which changes
after a predetermined, fixed time interval after said abrupt change
in said first signal; and
means responsive to said second output signal and operative upon
the occurrence of said change in said characteristic of said
control signal for determining the value of said second signal.
22. In a chemical analyzing system according to claim 21, the
improvement wherein said control signal is applied to said
differentiator circuit means for inhibiting the operation thereof
during said fixed time interval and wherein said determining means
measures the maximum value of said second signal.
23. In a chemical analyzing system according to claim 21, the
improvement wherein said determining means measures the
instantaneous value of said second signal upon termination of said
fixed time interval.
24. In a chemical analyzing system according to claim 21, the
improvement wherein said determining means measures the peak value
of said second signal after termination of said fixed time
interval.
25. In a chemical analyzing system according to claim 21, the
improvement further comprising:
means for displaying the determined value of said second
signal.
26. In a chemical analyzing system according to claim 21, the
improvement wherein said timing means produces a second control
signal, a characteristic of which changes before the termination of
said predetermined fixed time interval, wherein said second control
signal is applied to said differentiator circuit means for
inhibiting the operation thereof during a first portion of said
fixed time interval, and wherein said first-mentioned control
signal is applied to said determining means for inhibiting the
operation thereof during said fixed time interval.
27. In a chemical analyzing system according to claim 26, the
improvement wherein said determining means measures the
instantaneous value of said second signal upon termination of said
fixed time interval.
28. In a chemical analyzing system according to claim 26, the
improvement wherein said determining means measures the value of
said second signal at a known time after termination of said fixed
time interval.
29. In a chemical analyzing system according to claim 21, the
improvement wherein said sensor means comprises first and second
conductive elements for monitoring the conductance of said
solution, wherein said second electrical signal is proportional to
the rate of change of conductance of said solution, and wherein
said determining means measures the value of said rate of change of
conductance after said predetermined, fixed time interval from
introduction of said sample into said reagent.
30. In a chemical analyzing system according to claim 29, the
improvement wherein said conductive elements are substantially
planar and are positioned in said sample coplanar to minimize
capacitance effects on the measurement of conductance.
31. In a chemical analyzing system according to claim 29, the
improvement wherein said sensor means includes a surface which is
exposed to said solution, said surface conforming to a segment of a
sphere, and wherein said conductive elements comprise first and
second conductive areas positioned on said surface.
32. In a chemical analyzing system according to claim 31, the
improvement wherein said conductive areas have the shape of half
circles which are positioned with their straight sides parallel and
spaced apart.
Description
According to the present invention, a large, instantaneous change
in the characteristic of the solution being measured also takes
place when the sample is added. Therefore, means are provided for
measuring the value of the time derivative signal after a
predetermined, fixed time interval from introduction of the sample
into the reagent so as to eliminate the effect of the instantaneous
change in the characteristic of the solution and to permit thorough
mixing of the sample with the reagent.
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to a method and apparatus for
chemical analysis and, more particularly, to a method and apparatus
for the quantitative determination of the concentration of
substances which are reactive with enzymes.
Description of the Prior Art
One general area within the field of this invention is the chemical
analysis of biological substances to determine the chemical
composition thereof. For example, a common procedure is to
determine the concentration of glucose in blood or urine since the
concentration of glucose in these body fluids is indicative of the
operation of various fundamental body functions. Another common
procedure is to determine the concentration of urea in blood serum
since the concentration of urea in this body fluid is indicative of
the operation of the kidneys.
Most available analyzing systems for determining the chemical
composition of biological substances rely on colorimetric analysis.
For example, one known technique for the enzymatic assay of glucose
in blood and urine relies on the oxidation of the glucose in blood
with the enzyme glucose-oxidase to produce hydrogen peroxide and
gluconic acid. A presently available chemical analyzer relies on
the spectrophotometric response of the color reaction between
hydrogen peroxide, peroxidase and a chromogen. Another example
would be in the determination of urea in blood by the reaction of
urea with the enzyme urease to produce ammonium carbonate and using
colorimetric techniques for determining the intensity of the
product of the reaction.
While such colorimetric chemical analyzing systems are capable of
producing accurate indications of the concentration of a component
in a sample, there are several problems associated therewith. In
the first instance, most colorimetric techniques are subject to
large disturbances and interferences which may provide grossly
inaccurate indications. For example, in the enzymatic assay of
glucose in blood and urine by the oxidation of glucose with
glucose-oxidase, the strong oxidizing agent, hydrogen peroxide, can
react with other reducible substances and other impurities
interfere with the peroxide-peroxidase reaction causing a loss in
specificity and accuracy. In addition, availabe colorimetric and
analyzing systems require measurement of the intensity of the color
of the product at the completion of the reaction. Accordingly, the
analysis is time consuming. In addition, the assay often cannot be
conducted without deproteinization of the blood samples or
prepurification of urine samples.
With respect to some enzymatic reactions, it has been proposed to
use conductivity measurements in order to determine the
concentration of a component in a sample. More specifically, in
certain enzymatic reactions, a change occurs from a non-ionic to an
ionic species or from an ionic to a non-ionic species. In such
cases, the AC conductance of the medium serves as a direct measure
of the extent of the reaction and the rate of change of AC
conductance measures the rate of the reaction. Since the rate of
reaction is directly proportional to the concentrations of certain
reactants, such as the enzyme and the substrate, the concentrations
of these species can be monitored by measuring the rate of change
of AC conductance.
An example of this type of reaction is the reaction that occurs
when blood, containing urea, is mixed with the enzyme urease. The
non-ionic urea in the serum reacts with the enzyme urease to form
ionic ammonium carbonate. The rate at which ammonium carbonate is
formed is proportional to the quantity of urea in the serum sample.
Since ammonium carbonate is ionic, the AC conductivity of the
solution will change at a rate proportional to the quantity of urea
present.
U. S. Pat. No. 3,421,982 to F. C. Schultz et al for Enzymatic
Analysis proposes a system for measuring this change in AC
conductivity. The system of Schultz et al employs conventional
conductance electrodes and heretofore conventional levels of urease
to provide a constant rate of change of concentration with time.
Schultz et al indicate that the conversion of the substrate
proceeds for several minutes but that the rate of change of
conductivity in the first minute is essentially linear. These
conditions are required so that a two-point kinetic method can
meaningfully be used, with an adequate approximation to the rate of
reaction being determined by measuring the finite change in
conductance occurring over a fixed time interval within the linear
portion of the reaction. Mathematically, this is a measurement of
66 C/.DELTA.t where .DELTA.C is the change in conductance during
the fixed time interval, .DELTA.t, which is approximately one
minute. As a result, the system of Schultz et al is cumbersome,
time consuming and subject to inaccuracies.
One system for solving not only the problems inherent in
colorimetric analysis systems but also in the conductimetric system
of Schultz et al is disclosed in copending application Ser. No.
618,859, filed Feb. 27, 1967 in the name of James C. Sternberg for
Rate Sensing Batch Analyzer and assigned to Beckman Instruments,
Inc., the assignee of the present application. The analyzer
disclosed therein provides a convenient method for rapidly
determining quantitative information concerning a series of
chemical, and especially biological samples. That analyzer
determines the concentration of substances reactive with enzymes
rapidly and accurately and uses small sample sizes. The analyzer of
that application relies on the measurement of true instantaneous
rate of reaction at very early stages of the reaction, before much
reactant is consumed. The recorded rate signal results in a sharply
defined peak corresponding to apparent maximum rate which is
directly proportional to initial concentration. The apparent
maximum rate is obtained in a relatively short time interval, thus
saving analysis time and permitting more samples to be run in the
same time interval. As applied to the direct monitoring of oxygen
consumed in a glucose oxidase-glucose reaction, the invention does
not require preliminary purification or deproteinization of blood
or urine samples, gives highly accurate results on an absolute
basis and is insensitive to many impurities known to interfere with
many other analytical procedures.
While such Rate Sensing Batch Analyzer solves the problems of the
prior art discussed hereinbefore, it has been found that such
analyzer is not ideally suited for many enzyme reactions. For
example, the Sternberg analyzer determines the quantity of glucose
in blood or urine by using an oxygen sensor to measure the rate of
oxidation of glucose with glucose-oxidase to produce hydrogen
peroxide and gluconic acid. The reaction may be controlled so that
there is no initial change in oxygen level when the sample is
introduced into solution with the reagent. On the other hand, when
determining the concentration of urea in blood serum by reacting
the serum with urease to form ammonium carbonate, obviously an
oxygen sensor cannot be utilized to monitor the reaction. If a
conductivity sensor is utilized for measuring change in conductance
of the solution, problems are encountered in measuring the maximum
value of the time derivative of the output of the sensor, as taught
and claimed in said copending application. This is because blood
serum itself is conductive and there is a large conductivity change
in the solution when the serum is added to the reagent. This
instantaneous jump in solution conductivity generates an apparent
maximum value of time rate of change of conductivity which
approaches infinity such that a meaningful output cannot be
obtained.
SUMMARY OF THE INVENTION
According to the present invention, there is provided a method and
apparatus for chemical analysis which not only solves the problems
of the prior art solved by the before-mentioned Rate Sensing Batch
Analyzer, but is applicable to a wider variety of enzyme reactions.
The present method and apparatus is capable of rapidly determining
the concentration of a component in a sample, such as biological
fluids. The present apparatus is rapidly set up and put into
operation, makes the determinations rapidly and accurately, uses a
small sample size, and measures true concentration.
The present method and apparatus relies on the measurement of true
instantaneous rate at a very early stage of a reaction before the
reactant is consumed and, even with gaseous reactants, the
reactions can be open to the atmosphere since the indicative data
is collected before back diffusion of gas into the solution can
influence the results. The present method and apparatus recognizes
that introduction of the sample into solution with the reagent may
cause an instantaneous change in the characteristic of the solution
which is being measured. Accordingly, the present system inhibits
measurement of the rate of change signal during a predetermined,
fixed time interval starting with introduction of the sample into
the reagent, which time interval is sufficient to eliminate the
effect of the instantaneous change in the solution as well as to
permit thorough mixing of the sample with the reagent. Immediately
after the termination of the fixed time interval, the present
system measures the value of the rate of change of the
reaction.
Briefly, the present invention contemplates a method and apparatus
for determining the concentration of a component in a sample, i.e.
the concentration of urea in biological fluids, such as blood
serum, wherein the sample, upon being introduced into solution with
a reagent, reacts therewith, causing a continuing change in a
characteristic of the solution, and wherein the rate of the
reaction is indicative of the concentration of the component in the
sample. A sensor is provided for monitoring the characteristic of
the solution and for generating a first electrical output signal
proportional thereto. Differentiator circuit means are provided for
producing a second electrical signal proportional to the time
derivative of the first signal, the time derivative signal being
indicative of the concentration of the component in the sample.
According to the present invention, a large, instantaneous change
in the characteristic of the solution being measured also takes
place when the sample is added. Therefore, means are provided for
measuring the value of the time derivative signal after a
predetermined, fixed time interval from introduction of the sample
into the reagent so as to eliminate the effect of the instantaneous
change in the characteristic of the solution and to permit thorough
mixing of the sample with the reagent. Thus, the present invention
is capable of handling a large instantaneous change in the solution
which is of little or no interest followed by a smaller and slower
change which is indicative of the concentration of the component of
interest.
It is therefore an object of the present invention to provide a
novel method and apparatus for chemical analysis.
It is a further object of the present invention to provide a method
and apparatus for the quantitative determination of the
concentration of substances which are reactive with enzymes.
It is a still further object of the present invention to determine
the concentration of a substance reactive with an enzyme by
measuring the value of the rate of the reaction after a
predetermined, fixed time interval from introduction of the
substance into the enzyme.
It is another object of the present invention to provide a method
and apparatus for measuring the concentration of urea in blood
serum.
It is still another object of the present invention to measure
enzymatic reactions rapidly and accurately with a minimum sample
size.
Another object of the present invention is the provision of a novel
electrode for measuring AC conductance.
Still other objects, features and attendant advantages of the
present invention will become apparent to those skilled in the art
from a reading of the following detailed description of the
preferred embodiments constructed in accordance therewith, taken in
conjunction with the accompanying drawings, wherein like numerals
designate like parts in the several figures and wherein:
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph illustrating oxygen (O.sub.2) concentration as a
function of time (t) in a glucose oxidase-glucose reaction;
FIG. 2 is a graph illustrating the time derivative of oxygen
concentration (dO.sub.2 /dt) for reaction of FIG. 1;
FIG. 3 is a graph illustrating AC conductivity (C) as a function of
time (t) for certain enzyme reactions, such as a urea-urease
reaction;
FIGS. 4-6 are graphs illustrating the rate of change of AC
conductivity (dC/dt) versus time (t) for the graph of FIG. 3 and
showing three alternative methods for measuring the value of the
rate of change signal after a predetermined, fixed time interval
from introduction of the sample into the reagent;
FIG. 7 is a simplified block diagram showing a preferred embodiment
of apparatus constructed in accordance with the teachings of the
present invention;
FIG. 8 is a partial block diagram showing a possible modification
to the apparatus of FIG. 7;
FIG. 9 is a view, partly in section, showing a preferred embodiment
of sample cup for use in the apparatus of FIG. 7;
FIG. 10 is a side elevation view of a preferred embodiment of
sensor for use in the apparatus of FIG. 7; and
FIG. 11 is an end elevation view of the sensor of FIG. 10.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The beforementioned copending application of James C. Sternberg
discloses a convenient method for rapidly determining the
concentration of substances reactive with enzymes by measuring true
instantaneous rate of reaction at very early stages of the
reaction. As applied to the determination of the quantity of
glucose in blood or urine, such analyzer contemplates measurement
of the rate of oxidation of glucose with glucose-oxidase to produce
hydrogen peroxide and gluconic acid. Measurement is made by
positioning an oxygen sensor within a sample cup and measuring the
rate of change of oxygen concentration.
Referring now to the drawings and, more particularly, to FIG. 1
thereof, at a time t.sub.0, prior to introduction of the
glucose-containing sample into the oxygenated glucose oxidase, the
level of oxygen O.sub.2 may have a value O.sub.2 . Upon
introduction of the sample, at time t.sub.1, the level of oxygen
follows a curve 10 which decreases asymptotically. If the output of
the oxygen sensor is applied to a differentiating circuit, an
electrical signal may be derived which is the time derivative of
the oxygen concentration signal and thus proportional to the time
rate of change of concentration of oxygen. With reference to FIG.
2, curves 11, 12 and 13 represent three possible outputs of such
differentiating circuit. More specifically, upon differentiating
the output of the oxygen sensor, the time derivative increases to a
maximum value and then decreases as the rate of reaction decreases.
The maximum value of the output time rate of change signal is
directly proportional to the initial concentration of glucose and
provides a convenient, rapid and accurate output signal.
Many other enzymatic reactions are such that introduction of the
sample into solution with the reagent cuases no instantaneous
change in the characteristic of the solution being measured such
that the teachings of the Sternberg application are applicable. On
the other hand, in some enzymatic reactions, introduction of the
sample into solution with the reagent causes an instantaneous
change in a characteristic of the solution which it is desired to
measure. For example, it is possible to determine the concentration
of urea in blood serum by reacting the serum with the enzyme urease
to form ammonium carbonate. The rate at which ammonium carbonate is
formed is proportional to the quantity of urea in the serum sample.
Since the serum is initially non-ionic and since ammonium carbonate
is ionic, the AC conductivity of the solution will change at a rate
proportional to the quantity of urea present. However, problems are
encountered in measuring the maximum value of the output of an AC
conductance sensor since blood serum itself is conductive and there
is a large conductivity change in the solution when the serum is
added to the reagent. Referring now to FIG. 3, curve 15 shows the
change in AC conductance with time measured by a conductance sensor
positioned within a sample cup. At time t.sub.0, when the cup is
empty, the AC conductance has a value C.sub.0 =0. At time t.sub.1,
when the sample cup is filled with the enzyme urease, the AC
conductivity jumps to a value C.sub.1 becuase of the conductivity
of the reagent. At time t.sub.2, when the serum is introduced into
the sample cup, there is an immediate jump in conductivity to a
value C.sub.2 because of the conductivity of the blood serum.
Thereafter, the conductivity continues to increase asymptotically
to a maximum value C.sub.3, the change in conductivity from C.sub.2
to C.sub.3 being caused by the formation of ammonium carbonate.
Referring now to FIG. 4, becuase of the instantaneous jump in
solution conductivity at time t.sub.2, the rate of change of AC
conductivity dC/dt initially jumps to infinity, as shown by the
dashed curve 16. After time t.sub.2, dC/dt decreases asymptotically
along dashed curve 17. However, it will be appreciated by those
skilled in the art that this instantaneous jump in solution
conductivity, at t.sub.2, generates an apparent maximum value of
dC/dt which approaches infinity such that a system which measures
the maximum value of time rate of change of the sensor output is
incapable of providing a useful output signal.
Referring now to FIG. 7, a simplified block diagram of the present
method and apparatus for chemical analysis, generally designated
20, includes a sample cup 21 in which the enzyme reaction occurs.
Sample cup 21 may have any one of many known configurations and
includes means for permitting introduction of the reagent and the
sample as well as means for insuring thorough mixing of the
solution. A preferred embodiment of sample cup 21 will be described
hereinafter with reference to FIG. 9.
Extending into sample cup 21 is a sensor 22 for monitoring a
characteristic of the solution or a component or a product of the
reaction and for producing a first electrical output signal on a
line 23 proportional to such characteristic. Accordingly, sensor 22
may be any type of known sensor such as the oxygen sensor of the
before-mentioned copending application of Sternberg, such as a
spectrophotometric sensor or the like. According to the preferred
embodiment of the present invention, sensor 22 is a conductivity
sensor, of a type to be described more fully hereinafter with
regard to FIGS. 10 and 11, including first and second spaced
electrodes for sensing the AC conductance of the solution within
sample cup 21.
A constant amplitude AC voltage is applied to one electrode of
sensor 22 from an oscillator 24. The output of oscillator 24 may be
a symetrical wave of any shape, i.e. sinusoidal, square,
triangular, etc., having any desired frequency, depending on
circuit parameters, as will be explained more fully hereinafter.
The change in AC conductivity of the solution produces a change in
the current on line 23 connected to the other electrode of sensor
22, which current is reflected as an amplitude modulation of the
basic frequency signal from oscillator 24.
The amplitude modulated signal from sensor 22 is applied to a
demodulator 25 which produces a DC voltage on a line 26
proportional to the AC conductivity of the solution in cup 21. Line
26 is connected to one fixed terminal 27 of a switch 28 which
includes a moveable arm 29. Arm 29 is connected to a suitable
display device 30 such as a digital voltmeter. Accordingly, by
positioning arm 29 of switch 28 in contact with terminal 27, the DC
voltage proportional to the AC conductivity of the solution may be
directly read out on display 30. This signal should appear as curve
15 in FIG. 3. This feature permits monitoring of the actual AC
conductance of the solution in sample cup 21 to determine the
values of C.sub.0, C.sub.1, C.sub.2 and C.sub.3.
The DC voltage proportional to AC conductivity on line 26 is also
applied to a differentiator circuit 31 which is operative to
produce, on a line 32, a second electrical output signal
proportional to the time derivative of the AC conductivity signal
on line 26. Thus, the electrical signal on line 32 is proportional
to the time rate of change of AC conductance of the solution in
sample cup 21 and is directly proportional to the concentration of
the reactants in sample cup 21.
It can therefore be seen that apparatus 20 is useful in monitoring
a large class of enzymatic reactions, such as those where a change
occurs from a non-ionic to an ionic species, or vice-versa, as
described more fully in the before-mentioned copending application
of Sternberg. Such a reaction occurs when blood serum containing
urea is reacted with the enzyme urease to form ammonium carbonate.
As explained previously, since the urea is initially non-ionic and
since ammonium carbonate is ionic, the AC conductivity of the
solution will change, and at a rate proportional to the initial
concentration of urea.
Referring again to FIGS. 3 and 4, curve 15 shows the output of
demodulator 25 on line 26 as a function of time. At time t.sub.0,
with sample cup 21 empty, the conductance has a value C.sub.0 =0. A
measured volume of reagent, containing the enzyme urease, is
injected into sample cup 21 at time t.sub.1, completely immersing
sensor 22. When this occurs, the AC conductivity on line 26 jumps
to a value C.sub.1 because of the conductivity of the reagent. A
more elaborate discussion of the reagent will be provided
hereinafter. A very small volume of sample serum is then introduced
into sample cup 21 at time t.sub.2 and mixed with the reagent.
Accordingly, and as shown in FIG. 3, at time t.sub.2 there is an
immediate jump in conductivity to a value C.sub.2 because of the
conductivity of the blood serum. In addition, the non-ionic urea
reacts with the urease to form ammonium carbonate at a rate which
is proportional to the quantity of urea in the sample. Accordingly,
the conductivity continues to increase until a maximum value
C.sub.3 is reached.
Differentiator 31 provides an output voltage proportional to the
rate of change of AC conductivity. Becuase of the instantaneous
jump in solution conductivity at time t.sub.2, the rate of change
of conductivity initially jumps toward infinity (dotted curve 16),
preventing the measurement of maximum value of time rate of change.
However, according to the present invention, the output on line 26
from demodulator 25 is applied to a rate sensing circuit 35 which
senses the jump in conductivity when the serum sample is injected
and which generates an electrical signal on a line 36 indicative of
such jump. Alternatively, the output on line 26 from demodulator 25
may be applied to a conductivity level sensing circuit (not shown)
which would sense the jump in conductivity when the sample is
injected and which would also generate an electrical signal
indicative of such jump. In any event, the signal on line 36 is
applied to a time delay circuit 37 which generates, on a line 38, a
suitable electrical control signal, a characteristic of which
changes after a predetermined, fixed time interval. The length of
this fixed time interval is chosen based upon several
considerations. In the first instance, the time interval is
selected to be long enough to permit the transient from the jump in
conductivity to disappear sufficiently to make an accurate
measurement of rate of change of conductivity. The time interval is
also selected to permit elimination of other transients, such as
temperature upset and the like. Finally, the fixed time interval is
selected to be long enough to permit thorough mixing of the sample
serum with the reagent. In a preferred embodiment, as described
hereinafter, the change in characteristic of the output of time
delay 37 occurs approximately 12 seconds after sample
introduction.
In any event, according to a first embodiment of the present
invention, the output of time delay 37, on line 38, is applied to
differentiator 31 for inhibiting the operation thereof until the
end of the time interval, at time t.sub.3. At time t.sub.3, after
the termination of the time interval, and as shown in FIG. 4, the
output 41 of differentiator 31, on line 32, rises to the actual
signal level (dotted curve 17) and then falls with the reaction
rate. When this occurs, a signal peak 42 is obtained which is
proportional to the value of the rate of change signal after a
predetermined, fixed time interval from introduction of the sample
into the reagent and is, therefore, proportional to the urea
concentration in the sample. This output from differentiator 31, on
line 32, is applied to a rate measuring circuit 40 which, in this
embodiment, senses and holds peak value 42 and applies this value
as an output signal over a line 43 to a second fixed terminal 44 of
switch 28. Accordingly, by moving arm 29 of switch 28 into contact
with terminal 44, the peak signal from differentiator 31 may be
read out on display 30.
It will be appreciated by those skilled in the art that the
operation of time delay 37, differentiator 31 and rate measuring
circuit 40 just described is only one specific manner of
affectuating the broader teaching of the present invention, namely
measuring the value of the output of differentiator 31 after a
predetermined, fixed time interval from introduction of sample into
the reagent. In the embodiment shown in FIGS. 4 and 7, the control
signal from time delay 37, on line 38, is used to inhibit the
operation of differentiator circuit 31 until time t.sub.3 whereupon
rate measuring circuit 40 measures the maximum value of the signal
on line 32 immediately thereafter. Other techniques are obviously
possible. For example, with reference to FIGS. 5 and 8, rate
measuring circuit 40 may be in the nature of a sample and hold
circuit and the control signal from time delay 37, on line 38, may
be applied to rate measuring circuit 40 to select the time or times
for sampling the output of differentiator 31. More specifically,
time delay 37 may be operative to generate on a line 48, a second
electrical control signal, a characteristic of which changes at a
time t.sub.4 occurring after time t.sub.2 but prior to time
t.sub.3. As shown in FIG. 5, this second control signal on line 48
inhibits differentiator 31 from time t.sub.2 to time t.sub.4, to
prevent the disturbance of differentiator 31 in the presence of the
large jump in conductivity at time t.sub.2. When such transient has
been eliminated, the second control signal on line 48 permits
differentiator circuit 31 to begin operation so that the output
thereof rises along curve 49 until reaching the actual signal level
(dotted curve 17) and then falls with the reaction rate. However,
even though differentiator 31 is permitted to start operation at
time t.sub.4, it is still desirable to wait until time t.sub.3 to
measure the output of differentiator 31 so as to provide a
sufficient amount of time to eliminate the effects discussed
previously. Accordingly, the output of time delay 37 on line 38 is
applied to rate measuring circuit 40 which is activated at time
t.sub.3. Rate measuring circuit 40 measures the instantaneous value
50 of the output of differentiator circuit 31, at time t.sub.3, and
applies such value as an output signal to display 30 via switch 28.
According to another embodiment of the present invention, and as
shown in FIG. 6, rate measuring circuit 40 samples the value 51 of
the signal on line 32 at a time t.sub.5 so as to derive the value
of the signal on line 32 at a predetermined time t.sub.5, which
need not necessarily coincide with the apparent rate peak 50.
Referring now to FIG. 9, a preferred embodiment of sample cup
includes a cylindrical, hollow body 60, forming a chamber 59, the
bottom of which is tapered at 61. The apex of tapered section 61 is
connected to a vertical passageway 62 which is connected to a
horizontal passageway 63 extending entirely through body 60,
adjacent the bottom thereof. One end 64 of passageway 63 provides
an inlet for conducting reagent from a suitable source through
passageways 63 and 62 into chamber 59. The other end 65 of
passageway 63 provides a convenient location for emptying the
solution in cup 21. It will be apparent that exit 65 is blocked
during filling of cup 21 whereas inlet 64 is blocked during
draining of cup 21.
Body 60 is open at the upper end thereof, at 66, and may include a
suitable collar 67 if desired. The tip 68 of a pipette 69 is
adapted to be extended through the open upper end 66 of body 60 to
introduce a very small volume of sample, such as serum, into the
reagent in chamber 59. In order to insure thorough mixing of the
sample with the reagent in sample cup 21, sample cup 21 includes a
stirrer 70. Stirrer 70 should have a shape similar to that shown in
FIG. 9. To prevent the problem of coupling a drive element to
stirrer 70, stirrer 70 may be magnetized and may be driven by the
rotating magnetic force generated by a rotating drive magnet 71
connected by a shaft 72 to a motor 73. In order to support stirrer
70 for rotation within body 60 of cup 21, the lower end of stirrer
70 may be tapered, as at 74, at approximately the same angle as
tapered section 61 of chamber 59. By making stirrer 70 of some
suitable material, such as teflon, the tapered surfaces 74 and 61
provide adequate bearing surfaces for stirrer 70. A drainage
passage from cup 21 is provided by slots 74' in the bottom of
stirrer 70. A suitable type of stirrer is disclosed in U. S. Pat.
No. 3,591,309 issued to Robert A. Ray et al and assigned to Beckman
Instruments, Inc.
In operation, motor 73 is activated to rotate magnet 71 at any
desired speed whereby stirrer 70 follows such speed of rotation. A
measured amount of reagent is introduced into chamber 59 of cup 21
via inlet 64 and passageways 63 and 62. Thereafter, a small volume
of sample, such as serum, is introduced into sample cup 21 via
pipette 69 where it is mixed with the reagent due to the action of
stirrer 70.
As shown in FIG. 9, body 60 of sample cup 21 may include an opening
75 in the side thereof, which is partially threaded, at 76, for
receipt of sensor 22. Any suitable sensor having a pair of
electrodes may be used for measuring AC conductivity. For example,
the before-mentioned U. S. Pat. No. 3,421,982 to Shultz et al
teaches the use of a pair of parallel electrodes in a
conductimetric system. However, in accordance with the teachings of
the present invention, such electrodes should have a specific
construction in order to eliminate many problems that occur in
conductance measuring systems. However, before discussing in detail
the preferred embodiment of electrode constructed in accordance
with the present invention, the following discussion of the
problems involved is provided.
Classical conductance is defined as the reciprocal of the
electrical DC resistance and is represented by the equation:
C = 1/R
where C equals conductance and R equals DC resistance. However, the
polarization effects of DC systems have required that most
instruments use an AC voltage to measure this so-called
conductance. In fact, an AC system measures the reciprocal
impedance Z in accordance with the equation:
Z = (X.sub.c.sup.2 + R.sup.2).sup.1/2
where X.sub.c is the capacitive reactance due to ions in the
solution. Normally the capacitive reactance term X.sub.c remains
quite large until frequencies on the order of several megacycles
are reached. Since frequencies this high are not practical, most
conventional conductimeters have a capacitance balancing circuit
built in to accomodate this problem.
In accordance with the teachings of the present invention, it has
been determined that the reason for this large capacitive reactance
term is the use of conventional parallel-plate sensors. Referring
now to FIGS. 10 and 11, there is shown a preferred embodiment of
sensor 22 which substantially solves this problem. Sensor 22
includes an elongated cylindrical body 80 having a diameter equal
to the diameter of the opening 75 in body 60 of sample cup 21. The
forward end of body 80 may be threaded at 81 to engage with threads
76 in opening 75. Body 80 may also include a retaining nut 82 which
is adapted to be tightened against the outer surface of body 60 of
cup 21. Extending into sample cup 21 is a surface 83. Positioned on
surface 83 are first and second electrodes 84 and 85 which are
connected to leads 86 and 87, respectively, extending through body
80 of sensor 22. Leads 86 and 87 may be connected to oscillator 24
and demodulator 25, respectively.
According to the teachings of the present invention, the capacitive
reactance term may be minimized and, in effect, eliminated, by
making electrodes 84 and 85 planar and by positioning them
coplanar. By so positioning electrodes 84 and 85, the DC resistance
is uneffected but the capacitance is substantially minimized
thereby permitting the capacitive reactance term X.sub.c to become
very small at a much lower frequency.
Surface 83 may be a flat surface and electrodes 84 and 85 may be
conductive areas deposited thereon. As a practical matter, making
surface 83 flat and planar is not compatible with the cylindrical
configuration of the wall of chamber 59 and would prevent rapid and
thorough mixing of the solution therein and efficient drainage
thereof. Accordingly, and as shown in FIGS. 10 and 11, surface 83
is generally curved, having the shape of a segment of a sphere.
With such a configuration, electrodes 84 and 85 may be in the shape
of half circles positioned with the straight sides parallel and
spaced apart. With such a configuration, it has been found that the
capacitive reactance term X.sub.c in the reciprocal impedence Z is
effectively reduced to zero by increasing the frequency of
oscillator 24 to 10 kHz. Of course, the frequency at which the
reciprocal impedence plateaus is quite dependent upon the electrode
configuration and the 10 kHz value only corresponds to the
electrode configuration shown in FIGS. 9-11. In any event, with
such an electrode configuration, the AC impedance becomes a very
close approximation to the DC resistance and no capacitance
balancing circuit is required. The output of oscillator 24 may be
connected directly to one of leads 86 or 87 whereupon the other
lead provides the electrical signal on line 23 for direct
connection to demodulator 25.
As explained previously, one of the advantages common to the
present invention and the Rate Sensing Batch Analyzer of James C.
Sternberg is the provision of convenient methods for rapidly
determining quantitative information concerning biological samples.
The present analyzer, as well as that of Sternberg, relies on the
measurement of true instantaneous rate of reaction at a very early
stage of the reaction, before much reactant is consumed. By
obtaining this rate in a relatively short time interval, analysis
time is saved permitting more samples to be run in the same time
interval. Accordingly, as applied to the present invention, the
quantity of enzyme reagent used is large compared to usual methods
whereby the reaction proceeds at a relatively rapid rate. The
useful reaction is an approximately exponential change of
conductance, as shown in both FIGS. 1 and 3, having a typical time
constant of 20 seconds. This is diametrically opposed to the
teachings of Schultz et al who use such a small amount of reagent
that the reaction proceeds very slowly and can be stated to be
approximately linear.
Still another factor must be considered in making a conductivity
measurement as described herein. More specifically, in enzyme
reactions generally, the reagent is usually buffered with salts
which are highly conductive so that the pH of the solution remains
relatively constant as the reaction continues. This technique
permits the reaction rate to proceed at its maximum potential
value. With the present invention, it is apparent that this would
be objectionable since it is herein desired to allow the
conductivity of the solution to change, which change and the rate
thereof is measured to determine the concentration of one of the
components of the reaction. Accordingly, the present invention
contemplates starting with essentially pure urease, dissolved in
water, a low salt preparation having a relatively low initial
conductivity. Typically, before the sample is injected, the
conductance C.sub.1, as in FIG. 3, is about 20-25 percent of the
final conductance C.sub.3. Again, the concentration of enzyme
reagent relative to the usual concentration of enzyme is relatively
high. When the sample is injected at time t.sub.2, the conductance
jumps to C.sub.2 which will have a value in the vicinity of 80
percent of the final conductance C.sub.3. Of course, the initial
conductance C.sub.1 of the reagent may have a wide range of values
since there still will be a change of conductance during formation
of ammonium carbonate. However, because of the inherent difficulty
of measuring a small change in a large signal, it is desirable to
keep the initial concentration as small as possible.
It can therefore be seen that in accordance with the present
invention, there is provided a method and apparatus for chemical
anaylsis which not only solves the problems of the prior art solved
by the before-mentioned Rate Sensing Batch Analyzer, but is
applicable to a wider variety of enzyme reactions. The present
method and apparatus is capable of rapidly determining the
concentration of a component in a sample, such as the concentration
of components in biological fluids. The method and apparatus
represented by block diagram 20 may be rapidly set up and put into
operation to make quantitative determinations of true concentration
rapidly and accurately, using small sample sizes.
The present method and apparatus relies on the measurement of true
instantaneous rate at a very early stage of a reaction before the
reactant is consumed and during a non-linear portion of the
reaction. On the other hand, the present invention reconizes that
introduction of a sample into solution with a reagent may cause an
instantaneous change in the characteristic of the solution which is
being measured. Accordingly, the present system inhibits
measurement of the rate of change signal during a predetermined,
fixed time interval starting with introduction of the sample into
the reagent, which time interval is sufficient to eliminate the
effect of the instantaneous change in the solution as well as to
permit thorough mixing of the sample with the reagent. Immediately
after the termination of the fixed time interval, the present
invention contemplates measuring a value of the rate of change of
the reaction. Several specific embodiments have been described for
accomplishing this result. In addition, the present invention
contemplates a novel conductivity sensor for eliminating capacitive
influences in a conductance measurement.
While the invention has been described with respect to several
physical embodiments constructed in accordance therewith, it will
be apparent to those skilled in the art that various modifications
and improvements may be made without departing from the scope and
spirit of the invention. Accordingly, it is to be understood that
the invention is not to be limited by the specific illustrative
embodiments, but only by the scope of the appended claims.
* * * * *