U.S. patent number 3,847,486 [Application Number 05/260,560] was granted by the patent office on 1974-11-12 for automated spectrophotometer apparatus and computer system for simulataneous measurement of a plurality of kinetic reactions.
Invention is credited to William C. McCabe.
United States Patent |
3,847,486 |
McCabe |
November 12, 1974 |
AUTOMATED SPECTROPHOTOMETER APPARATUS AND COMPUTER SYSTEM FOR
SIMULATANEOUS MEASUREMENT OF A PLURALITY OF KINETIC REACTIONS
Abstract
A spectrophotometer apparatus for the automatic positioning of
multiple samples and sample blanks for measuring, for example,
double differential absorbance, that is, sample absorbance with
respect to both blank absorbance and time. The apparatus contains
appropriate electronic hardware, for example suitable electronic
computer and printout devices to generate a final digital printout
of multiple analytical reaction rate results.
Inventors: |
McCabe; William C. (Wichita,
KS) |
Family
ID: |
22989659 |
Appl.
No.: |
05/260,560 |
Filed: |
June 7, 1972 |
Current U.S.
Class: |
356/434; 356/39;
356/246; 356/408 |
Current CPC
Class: |
G01N
21/253 (20130101); G01N 21/272 (20130101) |
Current International
Class: |
G01N
21/27 (20060101); G01N 21/25 (20060101); G01n
021/22 () |
Field of
Search: |
;356/244,246,39,96,98,205 ;23/259 ;233/26,11 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
D D. McCraken, "A Guide To Fortran IV Programming," John Wiley
& Sons, New York, 1965. .
Gilford Instrument Labs, "General Catalog," Oberlin, Ohio (1968),
p. 11. .
Perkin; Elmer, Model 202 Catalog and Accessory Price List, (1968),
Norwalk, Conn. .
Gottschalk, Commissioner of patents, Benson et al., 175 USPQ673
(1972)..
|
Primary Examiner: Wibert; Ronald L.
Assistant Examiner: Godwin; Paul K.
Claims
I claim:
1. An apparatus for making automated kinetic measurements
comprising a standard light source, a standard light sensitive
detector and a cuvette wheel assembly disposed between said light
source and light sensitive detector, and mounted for rotation about
an axle assembly, said cuvette wheel assembly containing a
plurality of cuvettes containing the samples to be analyzed, means
for rotating the axle assembly, thereby providing the sequential
positioning of the samples in the light path of the light source,
an idler gear assembly associated with the axle assembly and in
synchronous phase with the cuvette wheel assembly, a "peak-picker"
means associated with the detector indicating when a sample is in
the proper position for recording transmittance readings received
by the detector and an electronic computer system associated with
the peak-picker means and the idler gear assembly for generating a
final digital printout of multiple analytical reaction rate
results.
2. The apparatus of claim 1, wherein the axle assembly is provided
with a standard axle drive gear and the idler gear assembly is
provided with a standard idler gear, said axle drive gear and said
idle gear being substantially the same size and being in engaging
relationship with each other.
3. The apparatus of claim 1, wherein the peak-picker means is an
interval timing device which forms part of the electronic computer
system, said interval timing device functioning to close the
circuit between the detector and the computer-system at constant
time intervals, .DELTA. T for recording signals received from the
detector.
4. The apparatus of claim 2 wherein the peak-picker means comprises
a plurality of trip-pins associated for rotation with the idler
gear assembly, each of said trip-pins corresponding to a sample or
blank cuvette and being adapted to sequentially engage a
micro-switch which momentarily closes the circuit between the
detector and the computer system for recording signals received
from the detector.
5. The apparatus of claim 1, wherein the peak-picker means is an
electronic switch which forms part of the electronic computer
system, said electronic switch functioning to close the circuit
between the detector and the computer system when a maximum signal
is received from the detector.
6. The apparatus of claim 1, wherein the computer system comprises
a signal generator which generates differential readings for each
revolution of the cuvette wheel, a storage channel for storing said
differential readings for the first of said revolutions, a double
differential generator for receiving the differential readings from
said storage channel and said signal generator corresponding to the
second revolution of the cuvette wheel, means for simultaneously
releasing said stored differential readings, means for converting
the double differential readings to a final digital printout of
multiple analytical reaction rate results and means for using the
differential readings corresponding to the second revolution of the
cuvette wheel with subsequent differential readings corresponding
to a third revolution of the cuvette wheel to generate another
digital printout, and means for continuing this process until the
desired number of analytical measurements have been made.
7. The apparatus of claim 6, wherein revolution indicator means are
associated with the idler gear assembly for indicating to the
computer system which differential readings are to be stored and
which differential readings are to be sent directly to the double
differential generator.
8. The apparatus of claim 2, wherein revolution indicator means are
associated with the idler gear assembly for indicating to the
computer system which differential readings are to be stored and
which differential readings are to be sent directly to the double
differential generator.
9. The apparatus of claim 7, wherein said revolution indicator
means comprises a trip pin on the idler gear assembly operatively
associated with a revolution indicator switch.
10. The apparatus of claim 1, wherein the computer system comprises
a signal generator which generates differential readings for each
revolution of the cuvette wheel, a storage channel for storing said
differential readings for the first of said revolutions, a double
differential generator for receiving the differential readings from
said storage channel and said signal generator corresponding to the
second revolution of the cuvette wheel, means for simultaneously
releasing said stored differential readings from said storage
channel to said generator thereby generating double differential
readings, means for converting the double differential readings to
a final digital printout of multiple analytical reaction rate
results and means for clearing the storage circuit.
11. The apparatus of claim 10, wherein the means for clearing the
storage circuit comprises an alternate revolution indicator means
associated with the idler gear assembly for indicating to the
computer system which differential readings are to be stored and
which differential readings are to be sent directly to the double
differential generator.
12. The apparatus of claim 1, wherein the cuvette wheel is provided
with a hollow axle-bearing-conduit system for the introduction and
removal of said thermostating liquid into and out of a cuvette
wheel.
13. The apparatus of claim 12, wherein thermostating fluid from a
standard recirculating liquid thermostating means enters the hollow
axle-bearing-conduit system through a first conduit means axially
communicating with the bottom end of the hollow axle-bearing system
for introducing a thermostating liquid thereto, second conduit
means provide communication between said hollow axle-bearing and
the hollow cuvette wheel assembly for introducing said
thermostating liquid thereto, third conduit means provide
communication between said hollow cuvette wheel and said hollow
axle-bearing system for removing said thermostating liquid from
said cuvette wheel and fourth conduit means axially communicating
with the top end of the hollow axle-bearing system for removing
said thermostating liquid therefrom back to the said standard
recirculating liquid thermostating means.
14. The apparatus of claim 1, wherein said apparatus is utilized
for standard clinical enzyme reactions.
15. A method for making automated kinetic measurements comprising
placing a plurality of samples (S) and blanks (B) to be analyzed in
a cuvette wheel, passing said samples and blanks in a circular path
sequentially through the light path of a spectrophotometer,
measuring the transmittance readings for each S vs B as they pass
through said light path and generating a plurality of (S-B)
differential readings for the first revolution of the cuvette
wheel, storing the (S-B) differential readings, generating a
plurality of (S-B) differential readings for the second revolution
of the cuvette wheel and generating double differential readings
.DELTA. (S-B) through the sequential release of said previously
stored (S-B) differential readings from said first revolution,
converting the .DELTA. (S-B) differential readings to a digital
concentration printout of multiple analytical reaction rate
results, using the (S-B) differential readings from the second
revolution of the cuvette wheel with the (S-B) differential
readings from the third revolution of the cuvette wheel to generate
new double differential readings and subsequent digital
concentration printouts, and continuing this procedure until the
desired number of analytical measurements have been made.
16. The method of claim 15, wherein said method is utilized for
standard clinical enzyme reactions.
17. The method of claim 15 wherein the signals for each of the
samples and blanks are recorded when the cuvette is rotated into
proper alignment with the optical axis of the spectrophotometer as
indicated by the momentary completion of an electrical circuit for
each of said samples and blanks in synchronism with the alignment
of said samples and blanks with the optical axis.
18. The method of claim 15, wherein the signals received from the
detector for each of the samples and blanks are recorded at
constant time intervals, .DELTA.T.
19. The method of claim 15, wherein the signals for each of the
samples and blanks are recorded when the cuvette is rotated into
proper alignment with the optical axis of the spectrophotometer as
indicated by a maximum signal received from the detector.
20. A method for making automated kinetic measurements comprising
placing a plurality of samples (S) and blanks (B) to be analyzed in
a cuvette wheel, passing said samples and blanks in a circular path
sequentially through the light path of a spectrophotometer,
measuring the transmittance readings for each S vs B as they pass
through said light path and generating a plurality of (S-B)
differential readings for the first revolution of the cuvette
wheel, storing the (S-B) differential readings, generating a
plurality of (S-B) differential readings for the second revolution
of the cuvette wheel and generating double differential readings
.DELTA. (S-B) through the sequential release of said previously
stored (S-B) differential readings from said first revolution,
converting the .DELTA. (S-B) differential readings to a digital
concentration printout of multiple analytical reaction rate
results, using the (S-B) differential readings from the third
revolution of the cuvette wheel with the (S-B) differential
readings from the fourth revolution of the cuvette wheel to
generate a second set of double differential readings and
subsequent digital concentration printouts, and continuing this
procedure until the desired number of analytical measurements have
been made.
Description
BACKGROUND OF THE INVENTION
The present invention relates to an improved method and apparatus
for determining multiple analytical reaction rate results,
particularly for clinical enzyme reactions. More particularly, the
present invention is directed to a method and apparatus for the
proper positioning of a plurality of samples and sample blanks for
simultaneously making a plurality of rate measurements on each
sample using a particular arrangement of electronic computer
hardware to produce a digital printout of the reaction rate
results.
In the area of clinical chemical analysis, much work has been done
toward achieving completely automatic analytical capabilities. One
of the areas of routine analytical measurement which has not yet
obtained a completely automated status with advantageous results is
enzyme rate analysis for heterogenous samples, such as for example,
blood serum. The instrumentation presently available, such as for
example, a centrifuge type analyzer, suffers from at least one of
the following limitations. Existing apparatus for measuring
clinical enzyme reactions do not perform analysis at a rate
(samples per hour) sufficient for routine patient screening needs.
Furthermore, the results produced by well known spectrophotometer
apparatus are subject to considerable error due to the following
factors: (a) not using reactions specific for particular enzyme
analysis, that is, many colorimetric procedures are utilized; (b)
secondary reactions are frequently present requiring sample blanks
for correction; (c) existing systems are not capable of readily
checking for linearity of reaction versus time; (d) the
spectrophotometer error is related to the extinction coefficient
used in making calculations; and (e) no adequate means is provided
for checking for inadequate substrates while the tests are being
performed.
The elimination of the above sources of error requires the use of a
precise spectrophotometer system capable of making accurate
measurements in the ultraviolet region of the spectrum, the ability
to include a sample blank in all measurements, the ability to make
simultaneous measurements on a large number of samples and blanks,
the ability to determine the linearity of the reactions and the
ability to measure the blank corrected final absorbance and final
blank absorbance.
There are three basic systems of positioning a plurality of samples
or samples and blanks in the light beam of a spectrophotometer or
discrete wavelength photometer. One of the systems is the flow-type
system wherein samples flow continuously through a flow-through
cuvette fixed in a light path. The flow system can be of the
continuous type, such as for example, that represented by the
Technicon autoanalyzer flow system or the start-stop type as
contained in the Gilford Model 3400 enzyme analyzer. The flow-type
system suffers from the following limitations: It has a relatively
slow rate of pumping fluids which limits the rate of sampling.
Also, a relatively large volume of sample is required together with
a micro cuvette. Furthermore, when using such a system it is very
difficult to make measurements versus time. The start-stop flow
system also suffers from many limitations including a relatively
slow rate of pumping fluids coupled with a start-stop requirement.
Thus, the sample rate limiting factors are the flow rate and the
length of time required for the stop phase of the cycle.
The second type of system for positioning a plurality of sample or
samples and blanks in the light beam of a spectrophotometer is a
system requiring a linear back-and-forth motion of discrete samples
in individual cuvettes. This system can also be either continuous
or of the start-stop type. An example of this type of system is the
Gilford Model 244 automatic cuvette positioner. This type of device
moves from cuvette number 1 to cuvette number 2 to etc., and then
back to cuvette number 1, with a start-stop cycle. The limitations
of this system are as follows: The space factor is a problem when
considering the total length of a holder which would be required
for testing 30 or more samples or a combination of 30 samples and
30 blanks which would require 60 cuvettes. Also, the time needed to
move the device from the last position to the first position would
introduce a sampling rate limiting factor. In addition, multiple
readings for each sample versus time would be required. A further
disadvantage is that the mechanism required for changing the
direction of the linear motion of the cuvette to give a relatively
slow movement in one direction and a very fast reverse movement in
the opposite direction, i.e., 30 to 60 times as fast depending upon
the number of samples and blanks being tested, to maintain the same
measuring sequence, would place sever mechanical strain on the
system and complicate the entire apparatus.
The third type of device for positioning a plurality of samples or
samples and blanks in the light path of a spectrophotometer is that
involving the circular motion of the cuvettes containing the
samples and blanks to be analyzed in a wheel assembly. This is the
type of motion utilized in the apparatus of the present invention.
The features of this type of system are as follows. Optimum single
measuring conditions are provided wherein only a minimum sample
volume is needed and wherein planar cuvette windows or curved
cuvette windows normal to the light at its point of incidence on
the cuvette window are used to prevent reflectance and refraction
artifacts. The circular motion of cuvettes in a wheel assembly also
enables optimum sample-blank differential measurements to be made
wherein a single optical system is utilized with only a very small
variable in time. Thus only a single monochromator (light source)
and a single photometer (detector) is utilized for making
sample-blank differential measurements. The circular motion of the
cuvette wheel assembly also provides a maximum rate of sequential
positioning of the cuvettes in the light path of the
spectrophotometer. There is no known linear motion device or flow
system which can move cuvettes containing samples to be measured
into and out of the light path as fast as the circular motion of
the cuvette wheel assembly. Finally, the circular motion of the
cuvette wheel assembly produces maximum sample positioning in a
minimum amount of space. Thus the circular motion of the cuvettes
in the wheel assembly produces optimum physical and theoretical
measuring conditions in a minimum amount of laboratory space at a
maximum rate of analysis.
SUMMARY OF THE INVENTION
An object of the present invention is to provide an improved method
and apparatus for making automated kinetic measurements for
clinical enzyme reactions.
Another object of the present invention is to determine the
linearity of the reaction by making a plurality of measurements on
each sample.
Still another object of the present invention is to provide an
improved spectrophotometer apparatus for the automatic positioning
of multiple samples and sample blanks for measuring for example,
double differential absorbance, that is, sample absorbance with
respect to both blank absorbance and time.
A further object of the present invention is to provide an improved
method and apparatus for measuring different types of reaction
rates during the same analytical run.
A still further object of the present invention is to provide an
improved method and apparatus for automatically substrating a blank
measurement during an analytical run which is essential for
clinical enzyme analysis if rapid and error-free results are to be
realized.
Still another object of the present invention is to provide an
improved method and apparatus for making multiple discrete
measurements on a plurality of samples and sample blanks to affect
simultaneous reaction rate analysis on all of the samples.
Another object of the present invention is to combine with the
above spectrophotometer apparatus, appropriate electronic hardware,
including electronic computer and printout devices to generate a
final digital printout of these simultaneous multiple kinetic
measurements for clinical enzyme reactions.
Other objects and further scope of applicability of the present
invention will become apparent from the detailed description given
hereinafter; it should be understood, however, that the detailed
description and specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
Pursuant to the present invention it has been found that the
above-mentioned disadvantages may be eliminated and a much improved
method and apparatus for making kinetic measurements for clinical
enzyme reactions may be obtained by utilizing a spectrophotometer
taking advantage of the circular motion of cuvettes containing
samples and blanks in a wheel assembly and utilizing a single
monochromator as a light source, a single photometer as a light
sensitive detector and an electronic system for generating a final
digital printout of the clinical enzyme reaction rate results.
According to the present invention, a cuvette wheel assembly
adapted for circular motion is utilized. The purpose of the cuvette
wheel assembly is to hold and sequentially position a number of
cuvettes containing samples and blanks to be analyzed in the light
path of a discrete wavelength or band-pass spectrophotometer. The
sequential positioning of the cuvettes in the light path is
obtained by rotating the circular cuvette wheel assembly about its
axis utilizing appropriate motor and gear means. The positioning of
the individual cuvettes in the light path must be controlled to
very close tolerance and this sequential positioning must be
coordinated with the proper use of said motor-gear drive system as
well as the use of an electronic computer adapted to monitor the
electrical output of the photometer.
The general, overall operation of the apparatus of the present
invention can be defined as follows. First of all, the cuvette
compartments of the cuvette wheel assembly are brought to assay
temperature by one of two thermostating systems which will be later
defined. The individual cuvettes are then filled with the assay
solutions and blank solutions at the assay temperature and loaded
into the cuvette compartments of the wheel. The cuvette position
number one, which is the first sample, is then moved to a position
indicated by a reference line, said position being at a point where
the cuvette is ready to pass through the light path of the
monochromator. The wheel revolution indicator knob is then turned
to position 1. After a preincubation-lag time, which is sometimes
necessary, the start bottom is pushed to begin the measuring
procedure. The pushing of the start button simultaneously starts
the motor which through a gear mechanism rotates both the cuvette
wheel and the idler gear assembly and activates the computer
analytical system. Thus the reading and calculating cycle is
started. The cuvette wheel first makes one complete rotation with
no printout, storing the sample vs blank differential readings,
hereinafter noted as (S-B). As the wheel starts its second
rotation, the idler gear assembly automatically positions the
revolution indicator switch and the computer and printer function
to print out the analytical result. On each additional or on each
alternate rotation of the cuvette wheel, depending on idler gear
assembly and computer data storage capabilities another set of data
is printed out. This is continued until, for example, four sets of
data (answers) for each of a plurality of samples are generated. In
the case where each rotation has been set to take 30 seconds at 2
r.p.m. four sets of answers will be generated in either 21/2
minutes or 4 minutes. In the case where the cuvette wheel is
designed to hold 30 samples, the corresponding rates of analysis
will be either 720 samples per hour or 450 samples per hour. The
pushing of the start button also activates a timer which
automatically stops the rotation of the cuvette wheel after either
the fifth revolution, which corresponds to 21/2 minutes of
operation or after the eighth revolution, which corresponds to 4
minutes of operation. If linearity of reaction rate has not been
achieved for all of the samples, the analysis can be readily
repeated by merely pushing the start button.
After an analysis has been completed, a check can be made for
"masked" elevated samples by switching from a concentration mode of
operation to an absorbance mode of operation and then pushing the
start button. This operation generates a set of thirty sample vs
blank differential absorbance readings in one rotation of the
wheel, which takes 30 seconds. If these readings do not match the
corresponding sample concentration readings, then the samples
should be repeated after first diluting them. In a similar manner,
high levels of NADH (coenzyme) "oxidase" can be checked by removing
the sample cuvettes and replacing them with their corresponding
cuvettes. Then, with the mode switch set for absorbance
measurements, the start button is pushed. The cuvette wheel, by
making one revolution, will generate a set of 30 sample-blank
absorbance readings. If any of these absorbance readings are well
below the majority of the others, indicating a low concentration of
NADH during the assay, the amount of NADH added to these samples is
doubled and their analysis is repeated. It is desirable to maintain
an optimum NADH concentration during the time of analysis for all
enzyme reactions requiring NADH for the analysis.
The above two checks coupled with the routine use of blanks and
controls as well as the use of multiple point kinetic method for
analysis, as defined by the present invention, substantially
eliminates all of the common causes of gross error frequently found
in all other presently used systems for clinical enzyme analysis.
The system as defined by the present invention not only gives about
a 5 to 10 fold increase in the rate of analysis over most systems
currently used for enzyme analysis but also is one of the only
systems presently available for semi-automated enzyme analysis
which produces results which are not subject to gross analytical
error.
The rate of rotation of the cuvette wheel is related to several
factors and must provide a balance between optical error and
variation of sample concentration. As the r.p.m. is decreased, the
ratio of optical error to total signal decreases. Any optical error
is due mainly to slight misalignment, which decreases as the rate
of rotation decreases. As the r.p.m. decreases, the absorbance for
a given measurement increases which produces a greater signal. The
limiting factors to consider when decreasing the r.p.m. to minimize
the measuring error are as follows. In determining the rate of
sample analysis, three readings determine a line and four readings
allows for one of the readings to be disregarded, for example the
first or last reading. At one r.p.m., it takes either one or two
minutes per result or four or eight minutes per assay, which is
equivalent to four results. Many reactions are linear for only
about two to four minutes, therefore all readings after three
minutes, for example, are suspect. This supports two r.p.m. rather
than one r.p.m., thereby giving a maximum assay time of four
minutes and at least 450 samples analyzed per hour which is
sufficient because a faster rate of analysis is not presently
required.
One of the features of the present invention is to provide the
necessary interfacing between the cuvette carrier wheel assembly
and the spectrophotometer. This interfacing can be achieved by the
use of a "peak-picker" mechanism. As each cuvette rotates into
proper alignment in the light path, the peak-picker means closes
the circuit from the photometer to the computer. Readings are
always taken as pairs, that is, (S-B).sub.1, (S-B).sub.2, etc..
Thus, a sample reading is taken and this reading is stored as the
cuvette wheel moves and then a blank reading, that is, the second
of the pair, is taken. Each pair of readings is used by the
computer to generate a set of differential values. The (S-B)
differentials for samples 1, 2, 3, etc., noted as (S-B).sub.1,
(S-B).sub.2, (S-B).sub.3, etc. are either stored or used to
caculate .DELTA. (S-B) differentials depending on the idler gear
assembly function. The ilder gear assembly can be used to indicate
to the computer which revolution the cuvette wheel is on, i.e.,
whether the wheel is on a (S-B) storage revolution or on a .DELTA.
(S-B) calculating revolution. The .DELTA. (S-B) calculating
revolution is also the revolution on which a set of results is
printed out.
There are at least three types of peak-picker means which can be
used for proper interfacing, i.e., for closing the circuit from the
photometer to the computer when a cuvette rotates into proper
alignment in the light path of the monochromator. One possible way
of using a peak-picker means for closing the circuit from the
photometer to the computer is through the use of an interval timing
device to close the circuit at constant time intervals, .DELTA. T.
This type of device is not shown in the drawings. It would function
as part of the computer means. A second peak-picker means is to use
the idler gear means to indicate when a cuvette is in the proper
position for taking transmittance readings. For example, the
computer takes readings from the photometers continuous output of
electrical signals only when a micro-switch is closed and it is
closed only when the cuvette is in proper alignment with the
optical axis as indicated by a trip-pin on the idler gear assembly.
A third peak-picker means would be an electronic switch peak
picker. In operation, the light generated from the monochromator is
continuous and the electrical output from the photometer is also
continuous with the change in signal from the photometer being
determined by variations in the amount of light reaching the
photometer. The amount of light reaching the photometer is
determined by the position of the cuvette carrier wheel relative to
the optical axis (light path). When each cuvette is positioned with
its optical face perpendicular to this imaginary line (optical
axis) the amount of light reaching the photometer will be at a
maximum. As the cuvette moves through this position, the light
reaching the photometer increases to this maximum and then
decreases. The decrease on either side of this maximum is the
result of light lost by reflection, defraction and increase
effective cuvette path length. This maximum signal can be used to
signal the computer to take a reading at this instant of time.
In a preferred embodiment of the present invention, an electronic
switch contained in the computer closes the circuit each time the
detector signal reaches a maximum. In this embodiment after the
first revolution, a trip-pin on the idler gear turns the revolution
indicator to its alternate position. This opens the (S-B).sub.I
(.sub.I corresponds to the first revolution) circuit to channel A
(see FIG. 3) and closes the (S-B).sub.II (.sub.II corresponds to
the second revolution) circuit to the .DELTA. (S-B) generator. This
activates the sequential release of previously stored (S-B).sub.I
signals from channel A to be used with each corresponding
successive (S-B).sub.II signal to generate the corresponding
.DELTA. (S-B) signal. The trip-pin also activates a timer which
turns the motor off after a time interval sufficient for four
revolutions of the cuvette wheel. Each (S-B).sub.II signal is
stored in channel A and the corresponding .DELTA. (S-B) signal is
converted to a digital concentration printout (answer). This
operation continues for three additional revolutions, after which
the motor is automatically shut off thereby stopping the analysis.
Thus, four "equivalent" answers have been generated for each sample
being analyzed. Thus, in the preferred embodiment, the second,
(S-B).sub.II reading is used with the first (S-B).sub.I reading
(which has been stored) to determine a .DELTA. (S-B) and then the
second (S-B).sub.II reading is substituted for the first
(S-B).sub.I reading in the storage circuit. The difference between
the above two signals or readings, .DELTA. (S-B) is used by the
computer to calculate a digital answer which is automatically
printed out.
In an alternative embodiment of the electronic system utilized in
the present invention, the wheel is rotated until an indicator
light, not shown in the drawing, corresponding to the first of two
coupled revolution lights and the reference lines are matched. This
is an indication that the double trip-pin on the idler gear has
positioned the revolution indicator switch to close the (S-B).sub.1
circuit to channel A. The motor-on switch is then pushed which
starts the rotation of the cuvette wheel thereby initiating the
assaying of the samples. This step also starts an interval timer
which automatically turns the motor off after a time interval
sufficient for eight revolutions of the wheel. As each cuvette
rotates into proper alignment in the light path of the
monochromator, a peak-picker means momentarily closes the circuit
from the photometer to the computer. After the first revolution,
the double trip-pin on the idler gear positions the revolution
indicating switch to open the (S-B).sub.I circuit and close the
(S-B).sub.II circuit to channel B (see FIG. 3a). At the same time,
the sequential release of previously stored (S-B).sub.I signals
from channel A is also activated to be used with each corresponding
successive (S-B).sub.II signal to generate the corresponding
.DELTA. (S-B) signal. The .DELTA. (S-B) signal is converted to a
digital concentration printout (answer). After the second
revolution, the double trip-pin on the idler gear assembly
positions the revolution indicator switch to open the (S-B).sub.II
circuit and close the (S-B).sub.I circuit. The stored (S-B).sub.I
signals are then used with the (S-B).sub.II signals as they are
generated to determine a .DELTA. (S-B) signal. In this operation
the storage circuit for each sample is cleared and the entire
operation is repeated to obtain a second set of answers. The entire
operation is repeated and the cycle continues for eight revolutions
at which time the motor is automatically shut off, thereby stopping
the analysis. Four "equivalent" answers have thus been generated
for each sample being analyzed. As will be readily recognized, the
only difference between the preferred and the alternative
embodiments of the electronic system, as described above, is a
factor of two in the rate of sample analysis.
In all probability, a combination of mechanical and electronic
devices discussed above will be necessary to realize the optimum
interfacing between the cuvette holder wheel and the
monochromator-photometer system.
If it is technically difficult to obtain the proper combination
using cuvettes with planar windows, cuvettes with curved windows
having a center of curvature which is coincident with the axis of
the cuvette carrier wheel can be used. In this case, a slight
uncertainty in the position of the wheel at the time of measurement
would not significantly affect the measurement. This would allow a
simple timing device in the computer coupled with a synchronous
motor-driven simple gear system to keep the wheel and
spectrophotometer in phase. There are two technical problems with
this type of system. First of all, it is technically more difficult
to produce optically accurate curved optical glass cuvette windows
than it is to produce planar cuvette windows. Furthermore, most
spectrophotometers would not have the light focused properly to
avoid reflectance and refraction losses at the curved surfaces of
the cuvettes. A lens system could be utilized to eliminate this
problem.
In positioning the cuvettes in the light path of the
spectrophotometer, it is essential that each cuvette, at the time
of measurement, is positioned such that all light striking the
optical surface (window) is normal to said surface at the point of
incidence. Two different means must be used depending upon whether
planar or curved surfaces (windows) are utilized. In the case of
cuvettes with planar windows, a horizontal plane bisecting the
wheel contains the optical axis which is perpendicular to the axis
of rotation of the wheel. Each rectangular cuvette chamber is thus
positioned in the wheel so that one of two adjacent and mutually
perpendicular sides of each chamber is parallel to a plane
containing a radius of the wheel and the axis of rotation of the
wheel. The cuvette can be automatically positioned in this chamber
through the use of springs which force the cuvette against said two
mutually perpendicular sides of the chamber. The light incident on
the cuvette optical surface (window) is collimated (focused at
infinity) and therefore is normal to the cuvette surface at the
point of incidence. In the case of cuvettes with curved windows, a
horizontal plane bisecting the wheel also contains the optical axis
and is also perpendicular to the axis of rotation of the wheel. The
curved optical surfaces of each cuvette are coincident with two
series of vertically displaced circles with fixed, but different
radii for each series with all circles being centered around the
axis of rotation of the wheel. The light incident on the cuvette
optical surface is focused on the axis of rotation of the wheel and
therefore is normal to this surface at the point of incidence. The
absolute rotational position of the wheel is not critical as long
as some portion of the optical surface is in the light path at the
time measurements are taken.
The fact that "double differential" measurements, that is, the
(S-B) differential and the differences in (S-B) differentials are
being made, reduce considerably the optical requirements for
matching the cuvette with the spectrophotometer. The accuracy of
any single transmittance reading is of only secondary importance
because it is only the differential readings that are used in the
analysis as defined by the present invention and any systemmatic
errors, for example, optical errors, will cancel themselves out and
thus not contribute in an adverse way to the final result. Because
of this feature, plastic cuvettes could be readily produced through
molding, rather than the more difficult to produce optical glass
cuvettes, thus eliminating the latter problem. Thus, either the
planar cuvette window or the curved cuvette window system could be
used equally well as far as the final results are concerned. From
the above discussion it is apparent that the three systems
discussed above, that is, the motor-gear drive system, the planar
or curved cuvette system and the electronic computer system have
some flexibility with the final choice of systems being based upon
technical considerations.
As is well known in the spectrophotometry field of technology, it
is imperative to maintain a constant environmental temperature for
the cuvettes throughout the entire period of the reaction rate
analysis. In one of the features of the present invention, the
cuvette wheel is provided with a hollow axle-bearing system for the
introduction and removal of a thermostating liquid into and out of
the cuvette wheel. Thus, the thermostatic liquid having a constant
temperature controlled outside of the spectrophotometer apparatus
is introduced into and removed from the cuvette compartment through
the axis of the cuvette wheel. Incidentally, because of the manner
in which the thermostating liquid is circulated in the cuvette
compartment around the cuvette, no thermostatic liquid is disposed
between the monochromator and the sample cuvette as well as between
the photometer and the sample cuvette.
In an alternative means for thermostating the cuvette carrier
wheel, a thermoelectric heating-cooling means could be used. For
example, the wheel could be made of metal with thermoelectric
heating-cooling elements provided therein. Electrical contacts
could then be made with an exterior power supply through the hollow
axle and bearing of the cuvette wheel. In such an arrangement,
spring loaded carbon tip contacts could be mounted in the bearing
with corresponding copper contact strips mounted in the axle
bearing seat.
To provide a substantially light-tight system from the
monochromator to the cuvette wheel and from the cuvette wheel to
the photometer a telescopic light pipe device has been found to be
particularly effective. This feature provides a light-tight
mechanical coupling of the stationary parts of the system, that is,
the monochromator and the photometer, with the constantly moving or
rotating part of the system, that is, the cuvette wheel. The light
pipe device of the present invention can be telescopically adjusted
between the monochromator and the photometer to provide a
substantially light-tight environment between these elements and
the rotating cuvette wheel. Alternatively, a large black box can be
utilized to enclose the entire system. One of the advantages of
using the box is that it could serve the dual function of not only
providing a light-tight system when the wheel is in use but could
function as a storage box for the wheel when it is not in use. The
box can be disposed upside down over the wheel assembly and
fastened permanently to the wheel support rack. When the wheel is
not in use the box and wheel assembly could be removed and the box
turned right side up. The lid to the box, with a handle attached
thereto, could then be attached thus making a convenient carrying
and storage case for the wheel assembly.
It should be pointed out that the rate of revolution of the cuvette
wheel is somewhat flexible as are the number of sample cuvettes in
the wheel and the number of revolutions of the wheel required for a
complete analysis of all of the samples. The conditions specified
above are essentially optimum for minimizing analytical error,
including a convenient number of samples for a single analysis and
a rate of analysis which is sufficient to meet the needs of a
primary user, that is, a clinical chemistry laboratory. Because the
reagent mixing step in the analytical procedure is not part of the
automated operation of the present invention, different types of
reaction rate measurements can be made during the same analytical
run. This is a particularly advantageous feature of the present
invention. The automated kinetic measuring spectrophotometer system
of the present invention is the only such known device which can
automatically subtract a sample blank during an analytical run.
This is an essential step for clinical enzyme analysis if both
rapid and error free results are to be realized.
DESCRIPTION OF THE DRAWINGS
The present invention will become more fully understood from the
detailed description given hereinbelow and the accompanying
drawings which are given by way of illustration only and thus are
not limitative of the present invention and wherein,
FIG. 1 shows a perspective view of the spectrophotometer apparatus
of the present invention including a monochromator, a photometer, a
cuvette wheel assembly, and an idler gear means;
FIG. 1a shows another embodiment of the idler gear means of the
present invention;
FIG. 2 shows a plan view of the cuvette wheel in conjunction with a
telescopic light pipe device, a monochromator and a photometer;
FIG. 2a shows a front view section of the individual cuvette
compartments in the cuvette wheel;
FIG. 2b shows a side view section of the individual cuvette
compartments in the cuvette wheel;
FIG. 3 shows a schematic illustration of the computer elements
starting from the point where the signals are taken from the
photometer;
FIG. 3a shows a schematic illustration of another embodiment of the
computer elements utilized in the present invention; and
FIG. 4 shows a schematic illustration of both a preferred
embodiment and an alternative embodiment of the computer function
of the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The spectrophotometer apparatus of the present invention according
to FIGS. 1 and 1a comprises a monochromator 1 (light source) a
photometer 2 (detector) and a cuvette wheel 3 provided with a
motor-gear-axle assembly. The cuvette wheel is mounted for rotation
about an axle assembly 4 between the monochromator and the
photometer. The axle assembly 4 is provided at its upper portion
with an axle drive gear 5. A synchronous motor 6, which is located
in the vicinity of the upper portion of the axle assembly is
provided with a motor drive gear 7 which is adapted to engage the
axle drive gear 5 for rotating the cuvette wheel. The axle drive
gear is also associated with an idler gear 8 of an idler gear
synchronizing mechanism. Because the idler gear engages the axle
drive gear, the idler gear remains in synchronous phase with the
cuvette wheel at all times and can be used together with other
associated elements to trip switches indicating to the electronic
components the relative position of the wheel and what electronic
function should be performed. The idler gear mechanism could
perform the following functions: It is adapted to indicate when a
cuvette is in the proper position for taking "transmittance
readings." For example, readings are taken from the photometer's
continuous output of electrical signals only when a given
micro-switch 9 is closed and it is closed only when the cuvette 10
is in proper alignment with the optical axis, as indicated by a
trip pin 11 disposed on the idler gear shaft 12 of the idler gear
8. Measurements are always taken as pairs, that is, (S-B).sub.1,
(S-B).sub.2, etc. The (S-B) difference readings are then
transformed into .DELTA. (S-B) readings using the computer methods
and devices discussed above and hereinafter. One electronic means
capable of handling the above pairs of readings is embodied in the
Gilford Model 2430 oscillating cell (S-B) system. This device takes
a sample reading, stores this reading, moves the cell holder
linearly and takes a blank reading. Both readings are taken as
"voltage" which is directly proportional to absorbance. The
difference between these measurements, i.e., (S-B) is determined
and in this case recorded on a chart recorder. The (S-B) difference
readings are then transformed into .DELTA. (S-B) readings as stated
above, using the computer methods and devices discussed above and
hereinafter.
The idler gear mechanism is provided at its upper end, that is, the
end opposite the idler gear, with a bearing sleeve 13 which is
adapted to receive the idler gear shaft 12. The idler gear is
constructed to be identical in size with the axle drive gear of the
axle assembly so that the rotation of the idler gear is synchronous
with the rotation of the cuvette wheel. The synchronous motor, the
axle for the cuvette wheel and the idler gear mechanism is
supported above the photometer by a support rack 14. The upper
portion of the support rack is provided with a motor support 15 for
supporting the synchronous motor, a top-axle bearing support 16 for
supporting the top portion of the axle for the cuvette wheel, an
idler gear shaft bearing support 17 for supporting the idler gear
mechanism and a revolution indicator support 18 for supporting the
revolution indicator. The lower portion of the axle for the cuvette
wheel is supported by a bottom axle bearing support 19 which is
adapted to be secured to the upper portion of the photometer. As
one of the possible arrangements of the support rack, right angle
elements of the support rack are provided at their opposite ends
with male and female end portions which provide for the ready
assembly and disassembly of the support rack about the
photometer.
In one of the advantageous features of the present invention, a
hollow axle 20 for the cuvette wheel is utilized, said hollow axle
being provided with upper and lower axle bearing means provided
with channels 21 which communicate with conduit means 22 and 23 for
introducing a thermostating liquid into and removing it from the
cuvette compartments of the cuvette wheel from a source outside of
the spectrophotometer. Thus a thermostating liquid which is
controlled to a desired temperature, depending upon the particular
samples being analyzed is introduced through the hollow axle of the
cuvette wheel via conduit means 22 to the cuvette compartment which
contains the individual cuvettes. The thermostating liquid is
removed from the cuvette compartment via conduit means 23 through
the hollow axle for the cuvette wheel and returned to the point
where the liquid is being controlled at a predetermined temperature
outside of the spectrophotometer. The cuvette wheel and the cuvette
axle are provided with additional support brackets 24 which further
stabilize the cuvette wheel axle with respect to the cuvette wheel.
The individual cuvette compartments which contain the cuvettes are
provided with a hinged cover to exclude extraneous light from the
cuvette compartments.
FIG. 1 shows a preferred embodiment of the idler gear mechanism of
the present invention wherein an automatic switch reset element is
associated with the idler gear shaft for resetting the switch after
the first revolution of the cuvette wheel. For this purpose
revolution indicating switch 25 is associated with a reset
mechanism 26. Manual lever 27 is also associated with the reset
mechanism for setting the switch in its proper first revolution
position. The revolution indicating switch 25 is supported on
support rack 14 by support frame 18.
FIG. 1a shows an alternative embodiment of the idler gear mechanism
of the present invention wherein the sample-blank cuvette position
indicator (trip pin) 28 is provided with a number of pins 11
corresponding to the number of sample-blank cuvettes disposed in
the cuvette wheel. Because the sample-blank cuvette position
indicator is attached to the idler gear shaft, it rotates with the
rotation of the cuvette wheel. As the sample disposed in the
cuvette wheel is rotated into light communication with the
monochromator and photometer, the indicator 11 makes contact with a
mechanical switch peak picker (micro-switch) 9 which enables the
signal of the sample to be recorded at a time T.sub.1. The peak
picker indicates to the computer that the signal of the next
cuvette which contains a blank be recorded at time T.sub.2. Through
the use of appropriate electronic components the difference (S-B)
can be readily computed and stored. The idler gear shaft is also
provided with a double trip pin element 29 which is associated with
an alternate revolution indicator switch 30.
FIG. 2 shows a plan view of the cuvette wheel of the present
invention showing the top of each of the cuvettes 10 disposed in
the cuvette compartments 31 of the cuvette wheel. FIG. 2a and 2b
show a front sectional view of the cuvette wheel at a plane
parallel to the wheel axis and a side sectional view of the
cuvettes disposed in the cuvette compartments, respectively. The
individual cuvette compartments 32 contain a hinged cover 33. The
individual cuvettes are held in position against the cuvette
compartment walls by spring means 34 and 35. As can be readily
seen, the thermostating liquid 36 is free to travel through the
cuvette compartments around the individual cuvettes. It should be
noted that the cuvette compartments are provided with a front and
back slit which is adapted to transmit the light from the
monochromator to the photometer. As can be readily seen, no
thermostating liquid is disposed between the monochromator and the
sample or between the photometer and the sample. This arrangement,
of course, eliminates any error which might be introduced by a
thermostating liquid interposed between the sample and these
elements.
FIG. 2 also shows a plan view of the
monochromator-photometer-cuvette wheel wherein a telescopic light
pipe 37 is disposed between the monochromator and the cuvette wheel
as well as between the photometer and the cuvette wheel as a stray
light trap. The telescopic light pipe prevents the light from the
monochromator from being lost to the environment and furthermore it
performs the same function with respect to the light received by
the photometer. The telescopic light pipe also prevents light
present in the environment from effecting sample measurements.
Because of the telescopic feature, the monochromator and the
photometer can be moved toward or away from the cuvette wheel. FIG.
2 further shows the use of felt pads 38 and a metal flange 39 to
prevent extraneous light from entering into the system. As can be
readily seen, the cuvette compartments are provided with slits on
the inside and the outside of the cuvette wheel adjacent to each
cuvette sample. Advantageously, the slit should be smaller than the
diameter of the light pipe.
FIG. 3 shows a schematic illustration of the computer elements
which can be utilized to produce a final answer from the
appropriate signal generated by the photometer. The continuous
electrical signal from the photometer is fed into the peak-picker
means of the computer. The peak-picker means picks the maximum
signal from the photometer for each of the samples being analyzed
at time T.sub.x and then the maximum signal of the blank being
analyzed at time T.sub.x plus .DELTA. T. The signals of the sample
S and the blank B are then fed into a signal generator which
produces the differential reading (S-B). From this point the
obtaining of a printout of the answer can follow one of two
courses. In the preferred embodiment of the present invention,
during the first revolution, the revolution indicator switch 25 is
manually set to its data (S-B) storage mode, i.e., the circuit to
channel A is closed and all (S-B) readings are stored in channel A.
After the first revolution a trip-pin on the idler gear
automatically turns the revolution indicator switch 25 to its
alternate position. This opens the (S-B).sub.I circuit to channel
A, and at the same time, the (S-B).sub.II circuit to the .DELTA.
(S-B) generator is closed. The differential reading (S-B).sub.II
represents that reading taken on the second revolution of the
cuvette wheel. As the differential readings (S-B).sub.II are
introduced into the .DELTA. (S-B) generator, the sequential release
of the previously stored (S-B).sub.I signals are activated from
channel A to be introduced into the .DELTA. (S-B) generator to be
used with each corresponding successive (S-B).sub.II signal to
generate the corresponding .DELTA. (S-B) signal. At the same time,
a timer which turns the motor off after a time interval sufficient
for four revolutions of the wheel is activated. The (S-B).sub.II
signal which was originally introduced into the .DELTA. (S-B)
generator is now stored in channel A to be used in subsequent
calculations and the .DELTA.(S-B) signal is converted to a digital
concentration printout (answer). This operation continues for three
additional revolutions, at which time the motor is automatically
shut off stopping the analysis. Four equivalent answers have thus
been generated for each sample being analyzed.
FIG. 3a represents a schematic of the computer elements utilized in
an alternative embodiment for obtaining a digital printout of the
answer. In this embodiment, the (S-B) differential reading is
stored in channel A and noted as (S-B).sub.I differential readings.
The differential readings stored in channel A is the direct result
of the closing of the (S-B).sub.I circuit to said channel as a
result of the operation of the double trip pin on the idler gear.
After the first revolution, the double trip pin on the idler gear
positions the switch to open the (S-B).sub.I circuit and close the
(S-B).sub.II circuit to channel B. At the same time it also
activates the sequential release of previously stored (S-B).sub.I
signals from channel A to be used with each corresponding
successive (S-B).sub.II signal to generate a corresponding .DELTA.
(S-B) signal. The .DELTA. (S-B) signal is then converted to a
digital concentration printout (answer). The difference between the
operation of the electronic elements of FIG. 3a when compared to
FIG. 3 is that the differential reading (S-B).sub.II which is used
to calculate a .DELTA. (S-B) signal is not restored in channel A so
that it can be used to calculate a new .DELTA. (S-B) with further
readings. Rather, after the printout of the .DELTA. (S-B) answer,
the (S-B).sub.II signal is removed from the computer system so that
in order to calculate a new .DELTA. (S-B) signal for subsequent
measurements, the (S-B).sub.III signals must be stored in channel
A. Thus, eight revolutions of the cuvette wheel are necessary to
generate four equivalent answers for each sample being analyzed.
This is to be compared with the preferred embodiment as shown in
FIG. 3 wherein only five revolutions of the cuvette wheel are
required to generate four equivalent answers for each sample being
analyzed.
FIG. 4 is a schematic drawing illustrating the computer function
described with respect to FIGS. 3 and 3a. The signals received from
the photometer in the first revolution are fed into a computer to
determine a set of (S-B) differentials which are stored. The second
set of (S-B) differentials generated on the second revolution of
the wheel are used along with the said previously stored (S-B)
differentials to generate a .DELTA. (S-B) differential. In the
preferred computer storage system the second set of (S-B)
differentials, i.e., (S-B).sub.II, remains stored to be used on the
third revolution to generate a second set of .DELTA. (S-B)
differentials. This is the storage system illustrated schematically
in FIG. 3. In the alternate storage system the second set of (S-B)
differentials is not stored and the entire process must be
repeated. This is the storage system illustrated schematically in
FIG. 3a. Independent of which computer storage system is utilized,
the .DELTA. (S-B) signal is subsequently introduced into a
log-digital converter, a concentration computer, and an amplifier
and finally a digital printout of the answer is obtained.
The invention being thus described, it will be obvious that the
same may be varied in many ways. Such variations are not to be
regarded as a departure from the spirit and scope of the invention,
and all such modifications as would be apparent to one skilled in
the art are intended to be included within the scope of the
following claims.
* * * * *