Note: Descriptions are shown in the official language in which they were submitted.
34(~8
BACKGROUND OF THE INVENTION
This invention relateq to a method for measuring a
soluble constituent in a material such as a biological fluid.
Moxe particularly, the invention provides a constituent-
measuring method in which the sample material being analyzed
is subjected to a constituent-manifesting chemical reaction
in an optically-thin layer of the reactants. The layer is
examined under electromagnetic radiation with sensing means
that responds in a linear manner to the concentration of a
selected constituent-manifesting product of the reaction.
The invention enables such measurements to be made
repeatedly with precision even when ~he spatial distributions
of the material being analyzed and of the reaction-producing
reagents over the thin layer both are not uniform and differ
from one another, provided that the distributions are the same
or successive samples. Moreover, the procedures of the
invention provide highly sensitive measurements of constituent
concentrations as contrasted to the prior art.
Prior art regarding the invention includes the teachings
in the U.S. Patents Nos.:
Yagoda 2,129,754 Natelson 3,036,893
Natelson 3,216,804 Natelson 3,219,416
Natelson 3,260,413 Natelson 3,261,668
Natelson 3,331,665 Natelson 3,368,872
Natelson 3,502,438 Rey 3,526,479
Findl 3,526,480 Fetter 3,552,925
An object of the invention is to provide an improved
method for measuring constituents in a biological fluid con-
tained in a fibrous cuvette.
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1 A further object is to provide a procedure for measuring
one or more soluble constituents in a fluid material contained
A ' in a fibrous eY~e~ and which provides higher sensitivity
than the prior art.
Another object is to provide a method of the above
character that can employ as the cuvette a fibrous sheet member
that is free of material-constraining structure, such as a disk
of confined size or constraining rings on the sheet member.
Another object of the invention is to provide a
method of the above character in which the cuvette medium can
have one or more test sites that are unbounded.
It is also an object of the invention to provide a
method of the above character capable of precise and accurate
constituent measurements.
A further object of the invention is to provide a
method of the above character that requires only a minute volume
of the sample material, typically less than 3~ microliters.
Another object of the invention is to provide a method
of the above character capable of providing analyses rapidly,
and further with minimal setup.
A ~urther object of the invention is to provide a method
of the above character that can perform both rate-reaction
measurements and end-point measurements.
Other objects of the invention will in part be obvious
and will in part appear hereinafter.
The invention accordingly comprises the several steps
and` the relation of one or more of such steps with respect to
each of the others thereof as exemplified in the procedures
hereinafter disclosed, and the scope of the invention is
indicated in the claims.
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SU~RY OF THE INVENTION
In brief, the invention provides an analysis procedure
i.n which reagents and a sample solution in an optically-thin layer
chemically react to produce a reaction product that is a known
measure of a constituent in the sample solution. Upon illumi-
nation of the layer with electromagnetic energy of a selected
wavelength, a radiation detector or other energy-sensing
means having a llnear response provides a linear measure of the
concentration of the constituent of interest.
The reagents and the sample solution are spatially
distributed so that, ideally, the amounts of the various
reactants are balanced with each other throughout the test area
for optimum chemical interaction to produce the reaction product
of interest. A fibrous sheet preferably is used to contain
.. the reactants and serve as the reaction cuvette With this
arrangement, the balanced di.stribution of reactants often
involves delivering the reactants to the fibrous cuvette in a
selected sequence, and not necessarily with uniform or identical
dis tr ibuti ons.
Also, in at least many instances of the practice of the
invention, the reaction sites do not require a confining structure
such as a Yagoda ring. Instead, the reaction site can be
unbounded.
The invention can be practiced with the apparatus des-
cribed in the commonly-assigned United States Patent No.3,844,717
entitled "Press For Progressive Compression Of Liquid-Bearing
Absorbent Article" of L. Sodic~son and F. Lim The
press structure of that patent can be used to
deliver a sample solution such as blood serum
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1 for analysis in accordance with the invention described herein
from a sample of whole blood. Further, the fluorometer
structure of the patent can also be used to advantage in
the practice of the invention described herein.
One advantage of performing chemical constituent analyses
in accordance with the invention is that it requires little time,
particularly as compared to prior techniques. Further, the
practice of the invention requires a minimal amount of equipment.
For example, in the lactate analysis of whole blood with a
fibrous cuvette already treated with analysis reagents as
described below, the desired serum of the blood sample can be
transferred to the cuvette by means of the ultrafiltration
press described in the patent identified above, and the
lactate analysis completed, in a matter of five minutes or so.
For a fuller understanding of the nature and object of
the invention, reference should be made to the following
detailed description.
D T~ILED DESCRIPTION
The invention typically is practiced by introducing
a liquid sample solution containing the material to be analyzed
as a solute to a selected site on a fibrous sheet that serves
as the analysis cuvette. Multiple chemical reagents are in-
troduced to the fibrous cuvette either prior or subsequent to
introduction of the sample solution. The sample solution and
each reagent are introduced to the fibrous cuvette so that each
has substantially the same spatial distribution of concentration
for every performance of a given test or analysis. That is, in
performing an analysis on multiple samples in accordance with
the invention, the spatial distribution of different samples
7~3
1 across the several fibrous cuvettes is the same, i.e. repeatable.
So also the spatial distribution of each reagent is the same for
testing each sample.
The cuvette contains the sample solution and the reagents
in an optically-thin layer. This makes it possible to measure
the constituent of interest by examination of the reaction
mixture with electromagnetic energy and to secure a response
that-is linearly related to the unknown constituent concentration.
- As used herein, the layer of sample solution and
reagents is considered to be optically thin when its transmission
of the incident and detected output measuring energies at
every point in the field of view of the measuring instrument
vary essentially linearly with concentration of the constituent
of interest up to the maximum concentration that is to be
measured.
The constituent-manifesting reaction product of
interest produced in the reaction mixture under the foregoing
conditions is preferably measured with a fluorometer or other
radiant-energy responsive instrument that has a linear response
to the concentration of the substance of interest.
More particularly, in the examples set forth below,
tl~e reaction mixture of the invention produces a fluorescent
reaction product with a concentration responsive to the con-
centration of the unknown substance being measured. Upon
illumination from the fluorometer source, at every point on
the cuvette in the fluorometer field of view the constituent-
measuring reaction product in the reaction mixture fluoresces
with an intensity that is linearly proportional to concentration
so long as the layer is optically thin. The fluorometer de-
tector has a linear response to this radiant fluorescence
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'1~34U~
1 from every point In its field of view and hence produces an
electrical signal that is a linear function of the product of the
fluorescent energy and the fluorometer sensitivity profile
integrated over the area of the cuvette surface within the
fluorometer field of view. The fluorometer sensitivity profile
is the spatial distribution of the instrument's response over
its field of view to fluorescence from its lamp. Hence the
profile is a composite of the spatial characteristics of the
fluorometer lamp and of the fluorometer detector.
As noted above, the United States Patent number
3,844,717 describes a fluorometer construction suitable for
use in practicing this invention. The fluorometer of
that patent has equal spectral angles for the incident energy
and for the measured fluorescent energy. This is desired to
provide the instrument with a composite, i.e. lamp and detector,
sensitivity profile which is symmetrical and approximately
uniform about the detector boresight axis, albeit with some
variation radially rom this axis, to measure all points in the
fluorometer field of view uniformly. ~hat is, with the fluoro-
meter angle o~ incidence equal to the angle of sensed radiation,
the total optical path from the fluorescent source of the
reaction layer and then to the fluorometer detector is
essentially uniform, at least to a first order, for all points
within the fluorometer field of view.
Alternative to a fluorometer, the invention can be
practiced with an oscillometer that measures the interaction of
reaction mixture components with an oscillating electromagnetic
field. For example, the cuvette sheet can be placed between
two flat electrodes to form in effect a flat-plate capacitor.
3~
The instrument is calibrated to measure the change in capacitance
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1 due to the reaction product of interest at a selected frequency.
The fibrous material that forms the reaction cuvette for
practice of the invention is preferably of fibers that are
inert to the sample solution and to the analysis reagents, and
which are non-absorbing and are transparent to the electro-
magnetic wavelengths involved in the measurement. However,
these requirements are not mandatory, rather they facilitate
factors such as calibration and measuring precision, and enhance
measuring sensitivity. By way of example, the invention has
been successfully practiced with fibers of cellulosic material
as well as of fiberglass.
Fibers of glass, i.e. fiberglass, tend however to be
sufficiently fragile so that they break upon being
compressed with the press described in the
patent noted above. Accordingly, where such a press or
other structure that subjects the fibrous material to stress
is to be employed, it is considered preferable that fragile
fibers not be used or at least that the fibrous sheet have a
mixture with less ragile, e.g. cellulosic, fibers.
For measurement of a rate reaction, the sheet of fibers
preferably has a fiber structure such that all liquid reagents
involved cease spreading within the sheet in a time that is
short compared to the time during which the reaction of interest
proceeds linearly. That is because the linear portion of the
reaction should proceed in large part after all the reactants
have ceased spreading due to capillary action of the cuvette
fibers. In the measurement of a typical rate reaction, it is
desirable that approximately three-quaters of the linear portion
of ths reaction time take place after significant spreading
ceases.
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1~4U(~78
1 With further regard to the spreading of fluid materials
within the fibrous cuvette, it is desired that all materials
move through the sheet at the same rate. Otherwise, the
distributions of various reactants tends to become unbalanced
at different locations within the cuvette area. In view of the
vastly different flow characteristics of materials typically
involved in constituent analyses, this objective often may not
be realized to the desired extent. However, it has been found
that disparate spreading of different materials can in many
10 instances be limited by applying the materials to the fibrous
cuvette in a selected sequence. In particular, it has been found
that the application of large-molecule reagents, with or without
drying, prior to the application of reagents of smaller molecules
substantially diminishes the spreading of the reagent applied
first by the latter-applied reagent. That is, it has been
found that where a solution of small-molecule constituents is
delivered ~o a fibrous cuvette followed by a solution of large
molecule constituents, the heavy molecule material tends to spread
and push the lighter molecule material out ahead of it with
the result that the small-molecule material is concentrated in
an annular ring outside of the area of maximum concentration of
the large molecule material. This condition is undesired and
results in significantly lesser measuring sensitivity than where
the procedure is reversed, as further detailed hereinafter.
As example of fibrous sheets suitable for the practice
o~ the invention, Schleicher & Schuell test papers Nos. 903,
903-C, 404, 410 and 25, and Whatman test papers GF/A, GF, B,
and G~/C have all been used successfully. In some instances
Yagoda-type s~ot confining rings have been found desirable.
In general, it appears that these rings are more desirable on
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)4~78
1 non-fiberglass papers and on thinner papers. In particular,
wax confining rings have been used to advantage with the
Schleicher & Schuell 903-C and 410 papers, whereas good results,
including high sensitivity, have been obtained with Schleicher
& Schuell 404 paper without confining rings.
The foregoing comments concerning the sequence of
applying reagents applies also to the addition of the sample
solution.
The term sensitivity is used in connection with this
invention to describe the magnitude of rate of change in sensed
energy, e.g~ fluorescence intensity, for a given concentration
of the constituent being measured.
EXAMPLE I
As a first example of the practice of the invention,
blood is tested to measure the concentration of glucose. The
analysis uses the known hexokinase reaction
HK
Glucose ~ ~TP ~ G-6-P + ADP
G6PDH
G-6-P ~ NADP~ 6 PG ~ NADPH -~ H
the conventional practice of which is described for example in
the brochure "Diagnostic Test Combinations, Operating Instructions"
distributed by Boehringer Mannheim Corporation.
In the simplest procedure, a solution containing all
necessary rea~ents is obtained by dissolving one Smith Kline
Eskalab*Glucose tablet in 0.5 milliliter of distilled water.
Ten lambda of the sample serum to be analyzed is deposited on
a fibrous sheet such as S&S No. 903-C. The sample is deposited
continuously, rather than drop-by-drop, at the center of the
reaction site, as with a pipette. This is followed by depositing
* Trade Mark
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1 ten lambda of the dissolved ~=~L~i solution in the same con-
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tinuous manner at the same spot. The order of addition is
critical, as the observed reaction rate is three times greater
in the above case than if the solution is added before the serum.
The reaction site is then monitored with a fluorometer
which illuminates the reaction site at 340 nanometers wavelength
O~ser~S
and~63~ the fluorescence with a detector responsive to the
460 nanometers wavelength of maximum NADPH emission. The
fluorometer output signal is measured during the linear portion
of the reaction. The measurement preferably is of the rate of
NADPH production rather than of the total amount of NADPH
production to facilitate the measurement of the fluorescence
from the NADPH separate from the background fluorescent
radiation from other materials at the test site as well as the
cuvette sheet itself. The latter radiation is essentially time
invariant, whereas the fluorescence from the NADPH increases
with the increase in NADPH production. Also, care is taken to
avoid contact of the fluorometer end window with the cuvette
and the reaction mixture to avoid variations that otherwise
arise in the detected fluorescence.
The foregoing glucose analysis has been performed
successfully with a variety of fibrous sheets for ~he cuvette,
including S&S 404, 595 and 25 papers, and the Whatman GF/A,
GF/B and GF/C papers.
EXAMPLE II
Blood serum is tested for lactate concentration with a
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1 pre-prepared fibrous cuvette, as o~ S&S gO3-C paper with a wax
ring about the test site. The cuvette is prepared by first
depositing, at the center of the test site, LDH in an ammonia
suspension, such as Sigma Type III-Beef Heart. This is followed
by deposition at the same point of ten lambda of a water solution
of fifteen milligrams of pyradine nucleotide (NAD) per
milliliter with a trace of surfactant such as Brij*.
Where fresh serum is to be tested, the pre-prepared
fibrous cuvette is first treated by depositing ten lambda of
glycine-hydrazine buffer at a pH of nine. This is followed
by the deposition of ten lambda of the serum sample.
It has been found to be important in the preparation
of the fibrous cuvette that the LDH solution be added prior to
the NAn solution, otherwise the lactate analysis occurs with
significantly lesser sensitivity. It is believed tha-t this is
because the relatively heavy LDH molecules establish a bond
with the cuvette fibers or otherwise resist spreading upon the
subse~uent addition oE the lighter NAD molecules so that the
two reagents largely occupy the same portion of the fibrous
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sheet. When the rea~ents are deposit~d in reverse order, it
is believed that the lighter NAD molecules are moved laterally
outward from theFoint of deposition by the heavier LDH molecules
with the result that the cuvette has a concentration of LDH
molecules within a predominantly annular concentration of NAD
molecules. The sensitivity is also observed to be diminished
when the se~uence of adding buffer and serum is reversed,
i.e. when the sample serum is adde,d before the buffer. (Note that
* Trade Mark - a series of polyoxyethylene fatty alcohol
derivatives made by Atlas Powder Co.
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4t3~7~3
1 the addition last of sample serum for the lactate analysis of
this example is opposite to the preferxed sequence for the
glucose analysis of Example I.)
EXAMPLE III
As a third example, a fibrous cuvette is again prepared
in the manner of Example II, but the serum to be tested is
available as a dried blot of whole blood carried on S&S 903-C
test paper. The paper containing the whole blood sample is
placed over the pre-prepared reagent sheet with an intervening
filter sheet, as described in the above-noted
patent, and placed in the press described in that
patent. Prior to closing the press, the blood stain is
reconstituted. Where the stain is fresh, this is done by the
addition of twenty lambda of saline (0.9 percent normal NaCl
solution). The press is then closed to transfer the serum
through the intervening filter sheet to be fibrous cuvette.
Alternatively, where the blood stain is not fresh, it is preferably
reconstituted by the addition of twenty lambda of water with a
trace of Brij or like surfactant, and then subjected to pressure
to transer it to the reaction cuvette.
In either case, the press is left closed for approximately
one minute. The press transfers a known portion of the lactate
in the sample to the reaction cuvette, but typically insufficient
liquid is transferred to provide sufficient molecular mobility
for the reaction to proceed. Accordingly, the sheet that
initially carried the blood sample and the intervening filter
sheet are stripped from the reagent sheet, and twenty lambda
of the glycine-hydrazine buffer (pH of nine) is applied to
wet the lowermost reaction sheet. Again, the rate of NADH
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production is monitored with a fluorometer as set forth above.
For all of the foregoing tests, the fluorometer field
of view is approximately a one square centimeter circular area
centered on the point at which the reagents are deposited on the
reaction cuvette.
EXAMPL~ IV
As an illustration of the application of the invention
- to a more complex chemical reaction for blood analysis, a
fibrous cuvette of S&S 903-C paper is prepared as follows to
test blood serum for the concentration of triglyceride. Again
the basic chemical reactions for this analysis are known, as
described for example in the above-noted brochure of Boehringer
Mannheim Corporation under the heading "Neutral Fat (Tri-
glycerides) and Glycerol". However, lipase is used to hydrolize
the neutral fat to glycerol.
The first step in the preparation of the fibrous
cuvette is to apply to the center of a reaction site on S&S
903-C paper twenty lambda of lipase (Schwartz-Mann No. 25) as
a continuous stream or single droplet and allow it to dry.
This is followed by the deposition at the same spot of twenty
lambda of the enzyme solution glycerokinase-glycerophosphate
dehydrogenose, and again the fibrous sheet is allowed to dry.
Thereafter, ten lambda of a co~factor solution containing NAD
and ATP, and the Mg+~ activator for glycerokinase and Ca +
activator for lipase in Brij water solution of at least three
milligrams per milliliter is deposited.
It should be noted that the three reagents so far
added to the fibrous sheet have been added in the order of
decreasing molecular weight. The remaining preparation of the
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~L~4(~(i'7E~
fibrous sheet is to increase the concentration of the combined
enzyme solution by successive depositions, so as not to unduly
spread the material but rather to concentrate it over a limited
area, e.g. one square centimeter, of the fibrous sheet.
Accordingly, before the co-factor solution dries, another ten
lambda of the enzyme solution is deposited on the sheet and
the sheet is then allowed to dry. Thereafter, ten lambda of
a glycine-hydrazine buffer of pH ~ is deposited on the sheet
at the center of the reaction site followed by another ten lambda
of the enzyme solution and another drying step. The fibrous
reaction sheet is now ready for use, and can be stored until
needed.
To make a triglyceride analysis with the reagent sheet
prepared in the foregoing manner, ten lambda of the glycine-
hydrazine buffer is first deposited on the sheet ~ollowed by
ten lambda of the blood serum being tested. The reaction sit~
is then examined with a fluorometer for the rate of NADH
production. A second test is performed with ~he same pre-
prepared reaction sheet in the same manner except that ten
lambda of saline are deposited in place of the sample serum,
and the rate of NADH production measured in the same manner.
The differenae between the two rates of NADH production, as
measured one with the sample and the other with saline, is
the desired measure of the triglyceride concentration. It is
believed that such a differential measurement is needed to attain
high accuracy due to impurities in the reagents and/or in the
fibrous sheet forming the cuvette, and hence with pure materials
the second, blank test can be eliminated.
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~4~1)7~3
Although the foregoing examples have involved analyses
in which NADH is produced, the invention can equally be used
in performing analyses in which a fluorophor is consumed, and
the rate of consumption is monitored.
It will thus be seen that the ob]ects set forth above,
among those made apparent from the preceding description, are
efficiently attained and, since certain changes may be made in
carrying out the above method without departing from the scope
10 '
of the invention, it is intended that all matter contai.ned in
the above description shall be interpreted as illustrative and
not in a limiting sense.
It is also to be understood that the follo~ing claims
are intended to cover all of the generic and specific features
of the invention harein described, and all statements of the scope
of the invention which, as a matter of language, might be said
to fall therebetween.
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