Note: Descriptions are shown in the official language in which they were submitted.
1~)493~6
1 BACKGROUND
This invention relates to a method and apparatus for
measuring a soluble constituent of a material such as a biological
fluid. More 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 or other distribution of the reactants, and
the reactants are carried in a porous or fibrous medium such as
paper. The layer is examined under electromagnetic radiation with
sensing means that responds in a known and preferably linear manner
to the concentration of a selected constituent-manifesting product
of the reaction. The invention also provides an instrument for
practicing the aforesaid method.
The invention enables the foregoing measurements to be
made repeatedly with precision even when the apparent background
absorbance or emittance of the porous or fibrous medium which
supports the reaction mixture is relatively high and varies signifi-
cantly from one test site to another, and when the spatial distri-
butions of the material being analyzed and of the reaction-producing
reagents are not all uniform and vary from one another, so that the
reaction product is not uniformly distributed. The technique yields
repeatable measurements provided that the relative distributions `
are the same for 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 3,331,665
Natelson 3,036,893 Natelson 3,368,872
,
''` ': ' '
. . .
10'~93g6
1 Natelson 3,216,804 Natelson 3,502,438
Natelson 3,219,416 Ray 3,526,479
Natelson 3,260,413 Findl 3,526,480
Natelson 3,261,668 Fetter 3,552,925
Accordingly, it is an object of the invention to provide
an improved method and apparatus for measuring constituents of a
biological fluid contained in a porous or fibrous medium. A more
particular object of this invention is to provide a method and
apparatus for monitoring a chemical reaction, and/or measuring a
reaction product, contained in a fibrous medium with minimal
dependence on the uniformity of the distribution of reactants.
A further object is to provide a procedure and instru-
ment for measuring one or more soluble constituents in a fluid
material contained in a porous or fibrous medium and which provides
higher sensitivity than the prior art.
Another object is to provide a method and apparatus of
the above character that can employ as the reaction and analysis
vessel a porous or fibrous sheet member that is free of material-
constraining structure, such as a disc of confined size or constrain-
ing rings on the sheet member.
Another object of the invention is to provide a methodand apparatus of the above character in which the porous or fibrous
analysis vessel can have one or more test sites that are unbounded.
It is also an object of the invention to provide a
method and apparatus of the above character capable of precise
and accurate constituent measurement.
It is a further object of the invention to attain the
foregoing measurement results with unbounded test sites and with
minimal loss of precision due to variations in the volumes of
the reactants added to the test site.
~' ;
10~9386
1 ~n additiona] object of the invention is to provide a
method and apparatus o~ the above character that requires only
a small volume of the original sample material, often less than
one microliter.
Another object of the invention is to provide a method
and apparatus of the above character capable of providing analyses
rapidly, and further with minimal setup.
A further object of the invention is to provide a method
and apparatus 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.
SUM ~ RY OF THE INVENTION
Although described with specific reference to practice
with porous sheets of fibrous structure, the invention can be
practiced with other porous materials. In particular, chemical
spot test analysis can be performed according to the invention on
a porous medium such as is used for electrophoresis, chromato-
graphy or filtration, and which (1) is chemically compatible with
the reagent system, (2) absorbs the fluids into its interstices,
and (3) has sufficiently high optical scattering and sufficiently
low optical absorption at the wavelength or wavelengths of interest
so that there are multiple scattering interactions of light with
the medium which cause the light to traverse the reaction product
held in its interstices with an effective path length greater
than the physical thickness of the medium. Illustrative examples
of such other media which can be used, preferably in sheet form,
include cellulose polyacetate membranes as marketed by the Gelman
Instrument Company under the registered trademark Sepraphore ;
cellulose nitrate microporous membrane filters as available from
the Sartorius Corporation under the -trademark SM11304 ;cellulose
* Trade Mark
~ - 3 -
,,p.;~.,i,
10493g6
1 acetate electrophoresis membranes as marketed by Instrumentation
Laboratory, Inc.; thin layer chromatography plates sold under
the trademark Avicel Uniplate available from Analtech Inc.
(microcrystalline cellulose media); and the silicic acid type
instant thin layer chromatography media available from the
Gelman Instrument Co. under the trade mark ITLC SA .
In brief, the invention provides an analysis procedure
in 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 of the sample solution. Upon
illumination of the layer with electromagnetic energy of a selected
wavelength, a radiation detector or other energy-sensing means
having a known and preferably linear response provides a linear
or other known measure of the concentration of the constituent
of interest. The reaction mixture is adjusted so that the concen-
tration of the reaction product at its most dense point is so
low that it absorbs only a small fraction of the radiation tra-
versing the fibrous medium.
The reagents and the sample solution are spatially dis-
tributed so that, ideally, the amounts of the various reactantsare balanced with each other throughout the test area for optimum
chemical interaction to produce the reaction product of interest.
* Trade Mark
- 3a -
.
~0~93~36
1 Preferred chemical s~stems produce rate reactions, and it is
desired to maintain optimal conditions of chemical balance across
the field of view of the measuring instrument. The fibrous sheet
contains the reactants for incubation and presentation to the
measuring instrument. With this arrangement, the balanced dis-
tribution of reactants often involves delivering the reagents to
the reaction site on the fibrous medium in a selected sequence,
and not necessarily with uniform or identical distributions.
This is done to overcome the chromatographic migration of re-
actants when subsequent solutions are added.
Also, in at least many instances of the practice ofthe invention, the reaction sites do not require a confining
structure such as a Yagoda ring. Instead, the reaction site can
be unbounded.
The invention also provides equipment for the preferred
practice of the foregoing procedure. In general, the equipment
provides self-adjusting and repeatable optical coupling from a
; radiation source to a radiation sensor by way of the reactant-
bearing fibrous medium. This coupling remains uniform over the
time course of the chemical reaction. To this end, the equipment
supports the fibrous sheet in an essentially fixéd geometry at
a fixed location. Also, the equipment retards evaporation of
liquid from the fibrous sheet, at least during the measuring
interval, and prevents undue compression of the reactant bearing
site.
In addition, the practice of the invention can employ
the apparatus described in U.S. Patent No. 3,844,717 issued
October 29, 1974, entitled "Press For Progressive Compression
Of Liquid-Bearing Absorbent Article" of L. Sodickson and F. Lim.
3~
-- 4 --
.. . .
~0493~36
1 The press structure of United States Patent No. 3,844,717
can be used to deliver a protein-free ultrafiltrate of a sample
solution such as blood serum or whole blood for analysis in accord-
ance with the invention described herein. Further, the fluoro-
meter structure of the copending application can be used to advan-
tage in the practice of the invention.
One advantage of performing chemical constituent analyses
in accordance with the invention is that it requires little time,
particularly as compared with 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
vessel already treated with analysis reagents as described below,
the desired serum ultrafiltrate of the blood sample can be trans-
ferred to the vessel by means of the ultrafiltration press
described in U.S. Patent 3,844,717 identified above, and the
entire lactate analysis from a finger puncture to analytical
result can be completed in a matter of three to five minutes.
Considered in further detail, an objective of this
invention is to provide a fast, simple, and low cost improved
chemical spot test or analysis, i.e. the measure of the production
of the product of a chemical reaction occurring in liquids con-
tained in a fibrous medium. The measure of reaction product can
be made either at the end of the reaction, i.e. an end point
measurement, or during the reaction, i.e. a rate reaction measure-
ment. Further, the invention is directed primarily but not
exclusively to measurements by means of radiant energy, i.e. by
photometric techniques in response to absorption or to fluorescence.
The photometric measurement of chemical reactions of
materials contained in a fibrous medium such as paper or a similar
fibrous sheet, has long been pursued because of the ease in hand-
ling such a medium and because of its low cost, particularly as
10493g6
1 a disposable medium for analysis. When used in conjunction with
the press of U.S. Patent 3,844,717, the present invention
allows the collection of specimens on filter paper and subsequent
analysis without need for centrifugation, as is commonly required
in the prior art.
However, several problems have made it difficult to
obtain the desired measurements with the required accuracy and
repeatability. One problem is that the paper or other medium
produces a high-level bac~ground photometric signal that varies
significantly from one reaction site to another, and even within
a site. In particular, investigators in the field of paper chro-
- matography have reported large variations in optical transmission,
optical reflectance, and native fluorescence from point to point
on analytical paper sheets. This location-dependent background
signal seriously degrades the precision of measurement of the
reaction-responsive signal of interest in chemical spot-test
analysis. Moreover, efforts to resolve this problem for paper
; chromatography have not been applicable to the somewhat different
art of chemical spot-test analysis.
In spite of this problem, the prior art as set forth
in the above-noted Natelson Patents Nos. 3,036,893 and 3,502,438
and the Findl et al Patent No. 3,526,480 teaches that the unknown
concentration of a chemical constituent in a specimen be determined
by comparing the observed optical measure developed on a reagent-
bearing site of a fibrous sheet exposed to the specimen of interest
with the corresponding optical measure of a similar site exposed
to calibration solutions of known concentration. The achievement
of repeatable and accurate quantitative results in this manner
presupposes that the measured difference in the optical property
at the several sites produced by the chemical reaction in propor-
tion to the desired constituent concentration be large compared
104938~;
1 to the background optical difference already pres~nt in the
fibrous s~eet at these sites.
The above-noted prior art also indicates that the non-
uniform distribution of reactants within a test site is a primary
source of error. This art accordingly teaches a variety of
techniques that are asserted to achieve precision by rendering
the distributions uniform within the instrument field of view.
To this end, U.S. Patent No. 3,036,893, for example, teaches
that a solution to be analyzed be distributed uniformly over a
reagent-bearing fibrous strip by applying the solution to the
strip through a porous screen. The '893 patent and U.S. Patent
No. 3,526,480 also teach the advantage of a uniform distribution
of reagents. However, these techniques remain subject to fluctu-
ations in the underlying medium itself.
These prior teachings describe the well-known "ringing"
phenomenon as a major limitation on the accuracy of quantitative
determinations performed on fibrous sheets. This occurs in the
prior art because the different concentrations of reaction
product present at different portions of the optical field of
view do not contribute proportionately to the measurement of
interest, whether it be optical transmission or reflectance, or
fluorescence.
Simply stated, the present invention recognizes that
the ringing phenomenon does not hamper the measurement in this
way if the ring itself absorbs little of the incident or re-emitted
light. Practice of the invention thus provides that all absorbing
molecules constituting the ring as well as those within the ring
be exposed to essentially the same incident light intensity and
do not attenuate the light emitted by neighboring molecules.
3~ However, reducing the concentration in the ring to this low a
value results in a chemically produced change in the optical
10493g6
1 ~easure which is small compared to background differences from
site to site. The prior art techniques were not satisfactory
at these low absorption levels.
The present invention circumvents both difficulties by
measuring the chemica~y produced change in optical property at
each site relative to the background at that same site prior to
the change-producing reaction. The resultant relative measure
of test sites is then compared with that of the calibration sites.
Thus, the present invention uses each reaction site as its own
blank for reference purposes. The resultant measurement is
essentially independent of the initial optical conditions of the
fibrous medium site, and hence is responsive to a maximal degree
only to the phenomenon of interest. The freedom from the adverse
effects of ringlng which result from this technique allows one
to measure the concentration of a reaction product resulting from
the direct deposition of a sample solution onto a reagent-bearing
fibrous medium, in direct contrast to the prior teachings noted
above. In particular, it has been found that an accurate measure
can be made of the product of a reaction taking place with re-
actants distribution non-uniformly within a fibrous medium when
the optical absorbance of the product is low, specifically well
below the 0.2 minimal level usually considered necessary for
meaningful measurements in classical in-vitro absorption spectro-
photometry. See for example, page 344 of Spectro-Chemical Methods
of Analysis, edited by J. D. Winefordner (Wiley-Interscience).
Moreover, the measure is essentially linear in terms of the con-
centration of the reaction product.
The foregoing condition of low optical absorbance is
referred to herein as an optically-thin condition. With re-
.
actants present in a manner that produces the reaction product
~ . .
. .,
10493~6
1 of interest in this low concentration level, the sensitivity of
the measurement to non-uniform distribution over the optical
field of view, and particularly to ringing, is rendered negligible.
In addition to the foregoing problems, it has been
found that the measurement of the optical response produced by
a reaction product in a fibrous medium varies significantly with
the degree of wetness of the medium and the degree of compression
of the reaction site. The problem with wetness is considered to
be unique to the present procedure, in which rate reactions are
monitored in progress and hence require liquid for molecular
mobility. This problem is avoided in the 3,036,893 patent, for
example, by drying the reaction site after reaction and prior to
optical measurement.
In brief then the invention provides apparatus and pro-
cedures for measuring the concentration of a selected constituent
of a fluid by means of a controlled chemical reaction that produces
a small modification to the optical properties of the highly
variable fibrous medium background. The measurement of interest
does not require uniform distribution of the reaction product ovex
the instrume~t field of view, but only a distribution that is
repeatable for different measurements. According to the invention,
the instrument and the procedure employ a stored or "remembered"
measure of the large, or apparently optically-thick, background
of the fibrous medium at the reaction site as a reference for
subsequent measurements made on the same site after initiation of
the optically-thin change of interest. The preferred practice of
the invention further provides for control of wetness and of fiber
compression at the reaction site. These techniques enable meaning-
ful measurements to be made even though the apparent absorbance
of the background medium ranges from l.0 when fully wet to l.7
when dry.
_ g _
104C~3~6
1 The invention also provides preferred techniques for
attaining repeatable constituent distributions within the reagent
pad or other fibrous medium, and for attaining a combination or
balance of constituents which yields a continuous high rate of
reaction at least for the desired measuring interval. This con-
tinuous high rate of reaction results in a corresponding high
sensitivity of the measurement. Further, the preferred techniques
described hereinafter for practicing the invention provide a re-
action which proceeds at a relatively uniform rate for a relatively
long time, which enhances measuring rate reactions with high sensi-
tivity.
The repeatable distribution is attained by the deposi-
tion of successive liquid reagents onto a fibrous medium in a
selected sequence to control and compensate for the washing out
of one reagent by successively applied reagents. Alternatively,
the concentrations of various reagents in a single solution are
adjusted to compensate for differences in their spreading or
affinity to the medium fibers when the solution is deposited on
the reaction site and the remaining procedure performed. Further,
the amounts of the several reagents are selected to yield a re-
latively optimum balance of all reacting constituents throughout
the test site. It has been found that the relative volumes needed
for this balanced condition are often different from those normally
used for attaining optimum reaction conditions in liquid mixtures,
i.e. in liquids contained in a vessel such as a test tube rather
than in a fibrous medium subject to chromatographic separation,
as is the practice of this invention.
With one preferred practice of the invention with un-
bounded test sites whose area exceeds the field of view of the
measuring system, the capillary properties of the pre-treated
~ .
-- 10 --
104g386
1 fibrous medium provide a buffer zone which can draw off an excess
of reagent or sample solution added to the reaction site. Under
selected conditions, the resultant measurement is primarily pro-
portional to the concentration of the unknown in the solution,
and relatively insensitive to the exact volume of sample or reagent
solutions added.
The invention accordingly comprises the several steps
and the relation of one or more of such steps with respect to each
of the others, and the apparatus embodying features of construction,
combinations of elements, and arrangements of parts which are
adapted to effect such steps, all as exemplified in the following
detailed disclosure, and the scope of the invention is indicated
in the claims.
BRIEF DESCRIPTION OF DRAWINGS
_____________________________
For a fuller understanding of the nature and objects of
the invention, reference should be made to the following detailed
description and the accompanying drawings, in which:
FIGURE 1 is a plot of the conventional characteristic
of optical transmission through a liquid as a function of optical
absorbance;
FIGURE 2 is a simplified showing of the practice of the
invention;
FIGURE 3 shows several plots of material concentration
and of the corresponding optical transmittance, both as a function
of spatial distribution;
FIGURE 4 is a perspective view, partly broken away, of
an instrument for practicing the invention;
FIGURE 5 is a fragmentary view of the instrument of
FIGURE 4 moved to the compression position;
.
~04938~i
1 FIGURE 6 is a fragmentary perspective view, partly
broken away, of a further instrument in accordance with the
invention;
FIGURE 7 is a cross-sectional view showing another con-
struction for the analysis instrument;
FIGURE 8 is a schematic diagram of the measuring
circuit according to the invention; and
FIGURE 8a is a schematic diagram of a configuration
similar to that in Fig. 8 but which uses a d.c. ratio circuit;
FIGURES 9 and 10 are graphs illustrating the practice
of the invention.
DESCRIPTION OF ILLUSTRATIVE AND PREFERRED EMBODIMENTS
The range of optical absorbance between approximately
0.2 and 0.8 which the prior art employs for chemical spot-test
analysis is shown in FIGURE 1 as being along section 12 of the
curve 10. The curve is a graphical representation of Beer's law
in that it plots percent optical transmittance through a liquid
as a function of the concentration of absorbing material in it.
The optical absorbance of the liquid is proportional to this
concentration, and in particular is the product of the total
number of molecules along the optical path o measurement and
the molar absorption coefficient of the molecules. Hence, FIGURE
1 also represents the relation between percentoptical transmit-
tance and concentration of a liquid. The transmittance varies
less linearly with absorbance, and with concentration, in the
curve section 12 than at the upper end of the curve, i.e. than
where the absorbance is less than 0.2.
Con~rary to the foregoing prior practice, the present
invention reveals the value of operating with a change in optical
absorbance due to the reaction of interest below that of the c~rve
section 12, and specifically in the "low absorbance end" section
14. This section corresponds with differential absorbance values
-12-
.~
~0493~6
1 of 0.2 and less, and preferably below 0.15, with a correspondingly
high percent transmittance of the reactant mixture on the fibrous
matrix. "Optically-thin" as the term is used in the specification
and claims refers to a condition wherein the concentration of
optically-active material participating in a reaction in a re-
acting mixture contained in a porous or fibrous medium is
sufficiently low that its absorbance of electromagnetic radiation
upon which measurements depend is less than 0.2 and preferably
below 0.15. In the case of measurements upon a fluorescent
radiation excited by interactions of an incident radiation with
the said mixture, the measurements depend on the absorbance of
both incident and fluorescent radiation being below the values
given above. The condition of greater optical density which the
curve 10 depicts in the section 12 and to the right thereof is
termed herein as being "optically thick".
FIGURES 2 depicts the practice of the invention with an
absorbance photometer 20. A sheet 16 of material of continuous
fibrous structure, e.g. paper, contains a liquid solution 18 of
chemical reactants. The reactants are selected to produce a
reaction product as a measure of a selected ingredient of the
initial reactants. An illuminating unit 20a of the absorbance
photometer 20 directs incident illumination, Io, on the solution
in the sheet 16. An optical detector and readout unit 2Ob of the
photometer, in optical alignment with the unit 20a and with the
sheet 16, receives the resultant illumination, I, which passes
through the solution-bearing sheet. The field of view 22 of the
- unit 20b at the sheet 16 is confined within the distribution or
spread of the solution 18 on the sheet, as shown. The wavelength
of the incident illumination, Io, and the wavelength of the re-
sultant illumination, I, to which the unit 20b responds are
,: I
~ ~ -13-
1049386
1 selected to provide in the unit 20b a measure which is primarily,
if not exclusively, responsive to the concentration of a selected
reaction product of the solution 18.
As indicated with dashed lines in FIGURE 2, the photo-
meter can be arranged to respond to the illumination which
reflects from the solution 18. That is, the illuminating unit
20a' and the readout unit 20b' can be arranged in optical
alignment, typically at equal angles of incidence and of
reflection, respectively,
-13a-
.
10493~
1 although other angles can be used depending on the difusion of
the illumination.
As FIGURE 2 further illustrates, the photometer 20 can
provide reflection and transmission measurements simultaneously
and with a single source with an arrangement employing the illu-
minating unit 20a', the reflection-sensing unit 20b' and a trans-
mission-sensing unit 20c' also in optical alignment with the
source unit 20a'. Similarly, the latter arrangement can provide
simultaneous measurements of fluorescence, with unit 20b', and
of absorbance, with unit 20c'.
The foregoing practice of the invention which FIGURE 2
illustrates is generally conventional. However, before describing
the invention further, the "optical thickness" of a solution
carried in a fibrous medium as this term is used herein will be
described with further reference to FIGURE 2.
The fibrous sheet 16 itself transmits only a small
fraction of the incident illumination, which illustratively has
a wavelength of 340 mm. The transmission is low primarily because
of scattering by the fibers of the paper sheet, rather than due
to absorption by them. In view of this phenomenon, the phrase
"attenuance" has been coined to describe the effect of the sheet
on the light passing through it, as distinguished from the usual
"absorbance". The term attenuance thus refers to the sum of
absorption by a material plus the removal of intensity from an
optical beam due to scattering.
The attenuance, as well as the reflectance and the
fluorescent emission, of the sheet 16 to the incident illumina-
tion has been found to have a strong dependence on the wetness
and on the compression of the sheet. This can be understood when
one considers the effects of liquid and of pressure on the fibers
- 14 -
(
1049386
1 and on the lnterstitial spaces. Paper scatters and reflects light
the most when it is dry because the interstices are occupied by
air, which has a refractive index that differs significantly
from that of the paper fibers. Water, however, is closer in re-
fractive index to that of the fibers, so that as water is depo-
sited on the sheet there is less scattering, lower reflectance,
higher apparent transmittance (i.e. reduced "attenuance"), and
reduced emitted fluorescence. This phenomenon is the basis for
the use of translucency agents in paper chromatography, whereby
fluids which match the refraction index of the paper fibers are
added to a dried chromatogram to render it as transparent as
possible for densitometry.
This theoretical model predicts that a beam of light
incident on the sheet 16 scatters within the sheet many times
before it is ultimately absorbed or emerges as reflected or
transmitted light. One therefore expects that the effective
path length of the light through the fibrous sheet is greater
than the thickness of the sheet itself.
This extended effective path length has been observed
in the course of analysis concerning the present invention. In
particular, this greater path length has been observed with the
apparatus described below with reference to FIGURE 7 by comparing
the observed fluorescence and transmission of paper discs wetted
with known volumes of NADH solutions having different concentra-
tions, with the corresponding fluorescence and transmission of
the same volumes of solutions in vitro, i.e. substituted for the
; paper disc. The apparent increase in effective path length has
in this manner been found to be between three and five.
It can thus be understood that an absorbing solution
placed on a fibrous medium such as paper can increase the apparent
~0493g6
1 "attenuance" of light by absorbing anywhere along the increased
path lenyth. If this absorption is followed by a fluorescent
re-emission, the fluorescent light must migrate through the scat-
tering fibers in the same ~lay as the exciting radiation, until
it emerges from the sheet or is internally absorbed. Further,
it is known, e.g. from U.S. Patent No. 2,554,321 of Bray, that
fluorescence measurements become non-linear if the concentration
of the fluorescing substance becomes so high that the incident
light beam is strongly attenuated in traversing the solution of
interest. The reason for the non-linearity is that the fluore-
scence-producing absorption by the outer fluid, which the illumin-
ation encounters first, reduces the contribution of inner fluid
to re-emitted light by decreasing the effective excitation inten-
sity in the inner fluid.
The reference to the "oPtical thickness" of the solution
in the present invention is based on the increased effective path
length described above.
In accordance with the invention, the optical absorbance
of the reaction product present in the solution 18 in the sheet
16, at the spectral range of interest, is maintained less than
0.2 and generally less than 0.15 for the effective path length
defined by the scattering within the sheet at the operating
wetness. That is, the effect of the solution on the sheet 16 is
optically thin at the spectral region of interest, at least within
the field of view 22 of the photometer unit 20b.
More particularly, FIGURE 3 shows the operation of the
FIGURE 2 instrument with different distributions of the measured
materials, i.e. the material constituents to which the photometer
responds. The prior art practice, noted above, prescribes that
these materials be distributed uniformly throughout the photometer
field of view. The graph of the spatial distribution of the measured
10493~6
1 materials in FIGURE 3A depicts such a uniform distribution. The
graph of FIGURE 3B shows the corresponding transmittance, i.e.
the ratio of resultant illumination I to the initial illumination
Io, of this distribution of the measured materials. The profile
of the latter graph is identical to that of the FIGURE 3A graph,
as expected.
~ IGURE 3C shows a non-uniform spatial distribution of
measured materials on the sheet 16 such as ideally results when
the materials are deposited on the sheet at the center of the
field of view and spread radially. The distribution has greater
concentration at the periphery of the resultant "spot" than at
the center, as is typically encountered with "ringing", i.e. the
washing out of liquid materials from the center of a spot towards
the periphery of the spot.
When the concentration of materials distributed per
FIGURE 3C is significantly optically thick, i.e. the optical
absorbance is far in excess o 0.2 (FIGURE 1), the optical trans-
mittance of the distribution has a profile such as the curve 24
of FIGURE 3D. This profile is not a linear response to the dis-
tribution profile of FIGURE 3C. Rather, it is highly non-uniform,
with less than proportionate transmittance from high-concentration
portions of the FIGURE 3C profile and with greater than propor-
tionate transmittance from the central, low-concentration portion
of the FIGURE 3C profile. It is because of these non-linearities
that the above-noted prior art prescribes uniform spatial dis-
tribution of the measured materials, per FIGURE 3A.
With a lesser concentration of the measured materials
distributed per FIGURE 3C, but still in excess of an optically-
thin absorbance, the resultant optical transmittance has a profile
as shown with the FIGURE 3C curve 26. This profile is still non-
linear relative to the FIGURE 3C concentration profile, but less
- 17 -
- : . .
10493~6
1 so than the grossly op-tically-thick condition of curve 24. By
way of illustrative example, where the peak-to-valley ratio of
the concentration curve in FIGURE 3C is two-to-one, the curve 28
for the optically thin condition should have the same peak-to-
valley ratio, but the curve 26 will have a lesser ratio, and the
ratio for the curve 24 will be even smaller, e.g. close to unity.
Hence, the FIGURE 2 instrument does not provide a linear
measure of the reaction product concentration when the materials
to which the measure responds are distributed non-uniformly in
an optically thick condition. Yet the importance of being able
to measure the concentration linearly becomes apparent from
examination of FIGURE 3E, which shows a concentration distribution
which is typical of that actually encountered in spot-test analysis
in the absence of the special procedure which the prior art teaches,
i.e. in the practice of the present invention.
However, an essentially linear measure does result when
the non-uniform distribution is optically thin. That is, when the
FIGURE 3C profile of spatial distribution of reaction product is
optically thin, i.e. has an optical absorbance less than 0.2 at
its most dense points, the corresponding proflle of transmittance
is a nearly linear response. The FIGURE 3D curve 28 depicts this
condition.
Thus the instrument of FIGURE 2 can provide a substan-
tially linear measure of concentration when the materials which
absorb the wavelength of interest are present in an optically-
thin layer or other condition. This finding remains valid even
in the presence of other strongly attenuating backgrounds, e.g.
due to the paper sheet 16 itself or in the initial reactant
solution, provided that the background attenuance is constant
during the interval of blank and signal measurements or varies
in a known and repeatable manner from sample to sample.
- 18 -
10493~6
1 Moreover, consistently precise measurements are realized
from different samples of the solution 18, on different sites of
the sheet 16, when the spatial distributions for the several
samples are the same, i.e. are repeatable. The prior art, by
contrast, considered that repeatable measures could be obtained
only with uniform material distributions.
FIGURES 4 and 5 show a fluorometer 30 with which the
invention can be practiced, rather than with an absorbance
instrument as previously described. The considerations regarding
an optically-thin condition and regarding repeatibility as pre-
viously set forth with reference to FIGURES 1 and 3 apply to a
fluorescence-responsive instrument. Moreover, a fluorometric
measurement yields greater sensitivity than an absorbance measure-
ment.
The illustrated fluorometer 30 of FIGURE 4 is combined
with a press 32 that transfers only the small-molecule fraction
of a sample liquid to a fibrous sheet, where that fraction under-
goes the chemical reaction desired for the measurement. The
compression of the fibrous sheet by the press usually is released
during the measurement. In particular, a dry, fibrous reagent
sheet 34 previously treated to bear the desired reactants is
layered under a further dry, fibrous, sample sheet 36 with an
ultrafiltration membrane or other filter 38 between them. The
liquid to be analyzed is deposited on the sample sheet, typically
with a pipette 40.
As described in full in the above-noted United States
Patent Number 3~844,717, when the press head 51 is moved in the
direction of the arrow from the position shown in FIGUgE 4 to
the position in FIGURE 5, it forces a significant portion of
the small-molecule fraction of interest of the sample from the
sheet 36 through the filter 38 to the reagent sheet 34. The
- 19 -
~049386
1 liquid fraction commences the des~red chemical reaction with the
reagents in the sheet 34. The fluorometer measures the reaction
product of interest, on either a continuous basis as appropriate
for a measurement of a rate reaction or after a selected time,
whichever is appropriate.
With further reference to FIGURES 4 and 5, the base 42
of the press forms a housing for the fluorometer 30. The fluoro-
meter illuminates the reagent sheet 34 with radiation from a lamp
44 through the platen 46 of the press; the platen is an optical
window transparent to the incident exciting radiation of the
lamp 44 and to the fluorescence with this radiation produces in
the reaction product of interest in the reagent sheet 34.
More particularly, the base 42 has a primary passage 48
and a secondary passage 50 therein; the passages are coplanar and
- are angled relative to each other with their central axes con-
verging at the sheet 34. The lamp 44 is mounted in the primary
passage 48 in optical alignment through the optical window of
the platen 46 with the section of the reagent sheet which is
centered under the press, i.e. which is under an inner foot
member 52 of the press. A primary filter 54 is mounted in the
primary passage interposed between the lamp 44 and the platen 46
to block unwanted radiation from illuminating the reaction pad.
Further, a reference detector 56, typically for producing an
electrical signal responsive to the intensity and the modulation
of the illumination from the lamp 44, is mounted in the primary
passage, preferably out of the optical path between the lamp and
the platen, as illustrated.
Although not required, the illustrated fluorometer
secondary passage 50 is oriented along the angle at which incident
radiation from lamp 44 reflects from the reagent sheet 34. Thus,
- 20 -
" . : ,
1049386
1 the illustrated fluorometer 30 has the secondary passage 50
aligned at the angle at which incident energy from lamp 44
reflects from the sheet 34 surface which is contiguous with
the optically-transparent platen 46. This geometry is preferred
to provide essentially equal-length optical paths, from lamp 44
to the detector 58 in the secondary passage 50, for illumination
impinging on all points of the reagent sheet 34 which are within
the fluorometer field of view. The equal-length optical paths,
in turn, result in high measuring accuracy and precision.
The secondary passage 50 mounts a fluorescence detector
58 and, in optical alignment between the detector 58 and the
reagent sheet 34, mounts a lens 60 which focuses the desired
fluorescence onto the detector 58. Also mounted in the secondary
passage are secondary filters 62, 64 and 66. These filters to-
gether block reflected primary radiation from the lamp 44 and
block background fluorescence and other radiation above and below
the pass band of the fluorescence to be measured.
Further, the secondary filters are selected to be non-
fluorescing, at least in the wavelength range of measurement,
when excited with reflected illumination from the lamp 44 and
with whatever fluorescence is present. Dielectric and metal
film filter constructions are preferred to provide the filters
62, 64 and 66 with the desired degree of non-fluorescence. This
provision of non-fluorescing secondary filters ln accordance with
the invention results in an increase in sensitivity and in back-
ground rejection of the fluorometer 30, as contrasted to a fluoro-
meter of similar construction except having glass or other
secondary filters which are known to produce broad band fluor-
escence encompassing the wavelength range of measurement.
Alternative to combining the press and photometer in a
single instrument as FIGURE 4 illustrates, they can be constructed
. . ~
10493~6
1 separate from each other, and used independent of one another.
For example, a sample liquid ready for analysis can be deposited
directly on a fibrous paper sheet and reacted with reagents, to
produce a reaction product which a fluorometer constructed as in
FIGURE 4 measures.
FIGURE 6 shows the details of a preferred construction
of a fluorometric instrument 70 for the practice of the invention.
~lthough described with reference to a fluorometric instrument,
the construction can be used with other forms of a photometer.
The optical elements of the instrument, i.e. the lamp, filters,
signal detector, and reference detector, can be arranged in
optical passages 70a and 70b as described above with reference
to FIGURE 4; the lamp and the signal detector are only indicated
schematically in FIGURE 6.
However, the support for the fibrous medium and the
exposure or optical coupling of the medium to the lamp and the
signal detector embody further features of the invention. More
particularly, the instrument 70 supports a fibrous medium illu-
strated as a paper strip 72 on a flat support surface 74a. A
reaction site 72a of the strip, which contains the reactant
mixture for producing the selected reaction product, is located
between an optical window 78, which is closely recessed by a
spacing 80 below the support surface, and a backing plate 82 `~
that extends laterally beyond the site to rest on the paper
strip above the support surface. An optically-transparent film
76 is wrapped around the strip 72. The film is contiguous with
the upper and lower surfaces of the strip and encloses the
reactant-bearing site 72a to prevent evaporation of liquid from
this site. The film 76 also precludes contamination of elements
of the instrument by the reactants of this strip. It hence
- 22 -
- ~ . . . : ~ .
10493~6
1 avoids the necessity of cleaning the optical window 78, the support
surface 74a and the backing plate 82 between measurements at
different reaction sites. The film 76 can be of an optically-
transparent, thermoplastic resin such as is produced by the poly-
merization of vinylidene chloride and is sold commercially under
the trade name Saran Wrap.
The housing 74 that forms the support surface 74a is
opaque to the radiation with which the instrument operates, and
the window 78 is transparent to the wavelengths of interest.
The window 78 is mounted in an aperture through the housing 74
and in optical alignment with the two optical passages 70a and
70b which illustratively are angled at 45 relative to one an-
other.
The backing plate 82 rests upon the paper strip 72
above the housing 84 and centered at the window 78. The illu-
strated backing plate 82 is hingedly mounted to the instrument
housing to facilitate lifting it clear of the support plate, i.e.
so that the backing plate is readily removed from, and alterna-
tively placed upon, the paper strip when access is desired to
the paper strip or to the support surface and the window. The
illustrated structure of the backing plate 82 is that of a disc
having a flat bottom surface. A stem 86 protrudes upward from
the back, upper side of the disc to a horizontal arm 88 that is
hinged to an upstanding post 90 on the housing. The backing
plate serves, by its slight weight, to hold the strip 72 flat
against the support surface 74a with the Saran or like film
pressed to the strip. This fixes the spacing of the reaction
site relative to the instrument source and signal detector.
Further, the plate, together with the wrap film 76, prevents
deformation of the strip at the reaction site due to the liquid
- 23 -
.. . .
10493~6
1 thereat. Although the strip can deform slightly into the spacing
80, such deformation is small due to the restraining effect of
the wrap film 76 and to the small depth of the spacing 80, which
preferably is less than the thickness of the strip. By way of
illustrative example, for a paper strip 0.017 inch thick, the
spacing 80 is 0.01 inch (approximately one-quarter millimeter).
One purpose of the spacing 80 is to allow the fibrous material
of the strip 72 to deform into it at the reaction site and thereby
avoid significant compression of the fibrous material under the
backing plate 82. It should be noted that the weight of the
backing plate on the strip is, however, slight.
With further reference to FIGURE 6, the backing plate
82 is sufficiently large to rest on the dry paper of the strip
72 which is located outward of the liquid-bearing and hence wet
site 72a. Hence, the thickness of the dry paper, which the plate
82 does not compress due to its limited weight, automatically
determines the spacing of the plate lower surface from the support
surface 74a. The plate 82 accordingly presses the upper surface
of the wet side 72a to be flat and hence coplanar with the upper
surface of the dry portion of the strip 72. As noted above, this
action can deform the wet site slightly into the spacing 80, which
is desirable because it allows the fibrous material at the reaction
site *o remain relatively uncompressed.
In the foregoing manner, it will be seen that the backing
plate 82 fixes the location of the reaction site relative to the
support surface 74a and hence relative to the photometer lamp and
signal detector, as is desired. Moreover, the backing plate pro~
vides this locating function automatically and with highly repeat-
able precision with automatic compensation for fluctuating thick-
ness of varying portions of the strip 72, and even from strip to
strip, because it is the dry portion of the strip surrounding the
reaction site which determines the spacing of the backing plate
above the support surface.
- 24 -
.. ,. ~
~049386
A further function of the backing plate 82 is that it
presents an optically-uniform surface behind the reaction site
72a. This surface can be highly reflective or highly a~sorbant,
although a reflective surface is generally desired to produce a
higher-level output fluorescent signal. The important factor is
that the surface be optically uniform throughout the instrument
field of view. The provision of optical uniformity also includes
avoiding air gaps between the strip 72 and the backing plate,
which the present structure attains.
The photometric instrument 70 of FIGURE 6 further in-
cludes thermal control elements for preventing fogging within
the photometer field of view due to moisture present within the
reaction site 72a. In particular, fogging of the upper surface
- of the window 78, and of the lower surface of the backing plate
82 has been observed with an instrument as shown in FIGURE 6 but
lacking thermal control. This fogging is understood to develop
when the lower surface of the backing plate 82 is at a different
temperature from the support surface 74a or the window 78, such
that liquid within the reaction site 72a vaporizes and condenses
on the cooler of the windor or backing plate surface. The fogging
is generally undesirable because it introduces a source of error
and imprecision to measurements. It has been found that the
fogging can be avoided by maintaining the backing plate 82 and
the window 78, together with the adjacent thermal mass of the
housing 74, at the same temperature. For this purpose, the
illustrated backing plate 82 has embedded therein a thermocouple
91, or other temperature sensor, and heating elements 92. The
illustrated temperature sensing element 91 is centered above the -
window 78 and hence the reaction site 72a. These thermal elements
are electrically connected to a regulated heating source. The
- 25 -
1049386
1 source can be constructed according to conventional techniques
to heat the backing plate to a selected, specified temperature.
Where desired however, the instrument can further include a
thermistor or other temperature-sensing element 93 disposed in
close thermal contact with the window 78, typically adjacent its
mounting in the housing 74. The sensor 93 is also connected with
the regulated source, which then energizes the heating elements
92 to maintain the backing plate at the same temperature as the
window 78.
An option~ element of the thermal structure of the
instrument 70 is a layer 84 of thermally-insulating material on
the underside of the backing plate 82. This layer is particularly
desirable where it is desired that the reaction site 72a equili-
brate thermally with the window 78, as when the backing plate is
made of metal or other thermally-conductive material and/or is ;~
not externally temperature controlled as by the heaters 91. The
layer 84 is readily provided with the desired uniform optical
property described above. In the illustrated embodiment, the
layer 84 is of a plastic consisting of tetrafluoroethylene poly-
mer such as that sold commercially under the name Teflon.
FIGURE 7 illustrates a further arrangement of a photo-
metric instrument according to the invention and for analysis of
a reaction product carried in a paper disc 94. In particular,
the instrument has a housing 96 fitted with an optical window 98.
The window is seated within a chemically-inert elastomeric gasket
100 that protrudes above the housing surface 96a to supportingly
- receive a carrier sheet 102 that is apertured to seatingly support
the disc 94. The aperture in the carrier sheet is sized suffi-
ciently close to the size of the paper disc to seat the disc with
a slight interference fit sufficient to carry the disc with the
' .
- 26 -
.: ... : .
10~9386
1 carrier sheet ~s considered preferable, the illustrated sheet
102 is slightly thinner than the dry paper from which the disc
is cut, e.g. a 0.015 inch thick carrier sheet for a 0.017 inch
thick dry disc. The carrier sheet is of optically-opaque material
and can be of metal shim stock.
The construction of FIGURE 7 can provide an essentially
vapor-tight space 106 between the disc 94 and the window 98. In
particular, the window upper surface preferably is recessed below
the surface 96a, as described above for the window 78 of the
FIGURE 6 instrument. The carrier sheet 102 is, as noted, seated
on an upper rim of the gasket 100, which protrudes above the
surface 96a and forms a seal therebetween.
The disc 94 can be enclosed with film such as the film
76 described above with reference to FIGURE 6 to prevent evapor-
ation from it. Alternatively it can be used without such a wrap,
and evaporation from it restricted to the small sealed space 106
between the carrier sheet and the window 98.
The illustrated photometric instrument of FIGURE 7 has
an optical window 104 overlying the disc 94 and the carrier sheet
102. The window 104 can be seated against the carrier sheet or
secured thereto, or spaced thereabove. With either construction
it provides an optical passage from the reactant-bearing disc 9~
to fu~ther optical apparatus (not shown) mounted within passages
108a and 108b of a further housing 108. The further optical
apparatus can, for example, include a fluorescence or an absorb-
ance detector where it is desired to measure the transmittance
properties of the disc 94. By way of illustrative example, the
arrangement of FIGURE 7 has been used with an optical source in
passage 96b, a fluorescence detector in passage 96c, and an
absorbance detector opposite the source in passage 108a. Alter-
natively, of course, the disc-supporting structure of FIGURE 7
- 27 -
104g3B6
can be used with an lnstrument as described above with reference
to FIGURE 6, and the second window 104 replaced by an opaque
backing plate.
As noted above, the invention resolves the point-to-
point variations in the optical properties of the reactant-
bearing fibrous medium by using each reaction site as its own
blank for reference purposes. That is, the same optical property
which is being used to measure the reaction product of interest
is measured for the fibrous medium at the reactant-bearing site
prior to production of that reaction product. This measure
identifies the desired optical property of the fibrous medium
site at which the reaction product of interest will be produced,
and with that site wet with all the reactants that are present
just prior to production of the constituent being measured.
~ccordingly, this measure identifies the background optical
parameter that is present at the reaction site when the reaction
of interest is occurring. Comparison of this measure with the
` same measure taken subsequently, i.e. during the reaction of
interest, produces a resultant signal that is responsive essen-
tially exclusively to the reaction product of interest. Hence,
- the resultant comparison signal has minimal dependence on the
optical background in the presence of which the product of
interest is being produced.
FIGURE 8 illustrates a preferred circuit for carrying
out the foregoing blanking procedure. A modulated supply 110
energizes a photometer source 112 with alternating voltage to
illuminate a sample 114, e.g~ the constituent of interest at a
fibrous reaction site, and illuminates a reference detector 116.
The lamp is modulated by the supply 110 at a frequency far removed
from sixty Hertz and harmonics thereof to preclude intereference
- 28 -
'' : :,
1049386
1 from power lines and li~e sources at that frequency. A signal
detector 118 responds to resultant output illumination from the
site to develop a sample-responsive signal, S, which it applies
to a ratio circuit 120. The ratio circuit also receives a
reference signal, R, which the detector 116 develops in response
to the lamp illumination. A synchronous demodulator 122 receives
the resultant ratio signal, which is proportional to the amplitude
ratio S/R and is free of fluctuations or other components due to
spurious variations in the source output illumination.
The demodulator, which is synchronized by the reference
signal, demodulates the alternating ratio signal to remove the
initial alternating modulation which supply 110 imparted to the
lamp illumination. The modulator output is thus a direct current
signal, which is applied to one input of a differential amplifier
126. The illustrated measuring circuit hence has the modulated
supply and the demodulator 122 to avoid sixty Hertz interference,
and employs a ratio circuit 120 to preclude errors due to lamp
fluctuations.
The FIGURE 8 circuit also has an adjustable voltage
divider 124 that applies a direct voltage of magnitude k, e~ual
to the value of S/R at time t = 0, i.e. prior to the development
of the reaction product of interest, to another input of the
differential amplifier 126. The response of the amplifier to
these signals is the desired difference signal, proportional to
(S/R - k), which is applied to a rate meter 130 for further pro-
cessing, recording or display as desired.
The voltage divider 124 and the differential amplifier
126 provide the above-noted blanking of the high background from
the sample bearing site 114. For this operation, the voltage
divider 124 is adjusted, either manually or with known automatic
- 29 -
104C~3~6
1 ~eedback techniques, to null the amplifier 126 output signal
prior to the measurement o~ interest. A preferred procedure
is to adjust the divider for null amplifier output during the
incubation period of the reaction of interest, i.e., immediately
after the reactant solution is developed at the reaction site
114, but prior to production of the constituent being monitored.
The voltage divider as thus adjusted served to store information
identifying the optical background of the site 114, and measured
with the same optical wavelength as the reaction product of
interest.
The measuring circuit of FIGURE 8 is illustrative of
many configurations which can, within the known skill of the
art, be constructed to provide the foregoing operation. For
example, FIGURE 8A shows a configuration that uses a d.c. ratio
circuit.
From the foregoing description it will be seen that
the invention typically is practiced by introducing a liquid
sample solution containing a material to be analyzed as a solute
to a selected site on a fibrous sheet that serves as the analysis
vessel, i.e. to contain the reaction mixture in the field of view
of the mea~suring instrument. Multiple chemical reagents are
introduced to the fibrous vessel either prior or subsequent to
introduction of the sample solution. The sample solution and
each reagent are introduced to the fibrous vessel 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
across the several fibrous vessels is the same, i.e. repeatable.
So also the spatial distribution of each reagent is the same for
testing each sample.
- 30 -
1049386
1 The fibrous vessel 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 essentially linearly related to the unknown
constituent concentration.
As stated above, the result of having an optically-thin
distribution of sample solution and reagents in the fibrous medium
is that the transmission of the incident energy, and of the de-
tected output measuring energy, at every point in the field ofview 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 concen-
tration of the substance of interest.
More particularly, in examples set forth below, the
reaction mixture of the invention produces a fluorescent reaction
product with a concentration responsive to the concentration of
the unknown substance being measured. Upon illumination from
the fluorometer source, at every point 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 over the
effective path length through the paper, i.e. fibrous medium~
The fluorometer detector has a linear response to this radiant
fluorescence from every point in its field of view and hence
produces an electrical signal that is a linear function of the
. :
- 31 -
10493g6
1 product of the fluorescent energy and the fluorometer sensitivity
profile integrated over the area of the fibrous surface within
the fluorometer field of view. The fluorometer sensitivity pro-
file is the spa~ial distribution of the instrument's response over
its field of view to fluorescence from its lamp. Hence the pro-
file is a composite of the spatial characteristics of the fluoro-
meter lamp and of the fluorometer detector.
As noted above, the fluorometer of FIGURE 4, as well
as the instruments of FIGURES 6 and 7, 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 boresiqht axis, albeit
with some variation radially from this axis, to measure all points
in the fluorometer field of view uniformly. That is, with the
fluorometer angle of incidence equal to the angle of sensed ra-
diation, the total optical path from the fluorescent source of
the reaction layer and then to the fluorometer detector is essen- -
tially uniform, at least to a first order, for all points within
the fluorometer field of view.
Alternative to either an absorbance photometer or 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
fibrous sheet can be placed between two flat electrodes to form
in effect a flat-plate capacitor. The instrument is calibrated
to measure the change in capacitance due to the reaction product
of interest at a selected frequency.
The fibrous material that contains the reaction vessel
for practice of the invention is preferably of fibers that are
~, . . .
~049386
1 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 'he measurement. ~owever,
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 successively 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 U.S. Patent 3,844,717 noted above.
Accordingly, where such a press or other structure that subjects
the fibrous material to stress is to be employed, it is con-
sidered preferable that fragile fibers not be used or at least
that the fibrous sheet have a mixture with less fragile, 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 pro-
ceeds linearly. This is because the linear portion of the re-
action should proceed in large part after all the reactants have
ceased spreading due to capillary action of the fibers.
In the measurement of a typical rate reaction, it is desirable
that approximately three-quarters of the linear portion of the
reaction time take place after significant spreading ceases.
With further regard to the spreading of fluid materials
within the fibrous vessel it is desired that all materials move
through the sheet at the same rate. Otherwise, the distri-
butions of various reactants tend to become unbalanced at dif-
ferent locations within the reaction and analysis site. In
10~93~36
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 disparet spreading of different materials can in many
instances be limited by applying the materials to the fibrous
vessel 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
to a fibrous vessel 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 pro-
cedure is reversed, as further detailed hereinafter.
As examples of fibrous sheets suitable for the practice
of the invention, Schleicher & Schuell test papers Nos. 903, 903-C~
404, 410 and 25, Whatman test papers GF/A, GF, B, and GF/C have
all been used successfully. In some instances Yagoda-type spot
confining rings have been found desirable. In general, it appears
that these rings are more desirable on 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 con-
fining rings.
- 34 -
.
~049386
1 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 1
As a first example of the practice of the invention,
blood serum is tested to measure the concentration of glucose.
The analysis uses the known hexokinase reaction
Glucose + ATP HK ~- G-6-P + ADP
G-6-P + NADP G6PDH> 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.
EXAMPLE la
In the simplest procedure, a solution containing all
necessary reagents is obtained by dissolving one Smith Kline
Eskalab Glucose tablet in 0.5 milliliter of distilled water.
Ten lambda of an appropriate dilution of the sample serum to be
; analyzed is deposited on a dry 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, without drying, by depositing ten lambda of
the dissolved Eskalab solution in the same continuous manner at
the same spot. The order of addition is critical, as the observed
initial reaction rate is three times greater in the above case
than if the solution is added before the serum.
. . .
:
1049386
1 The reaction site is then monitored with a fluorometer,
such as the FIGURE 4 fluorometer 30, which illuminates the re-
action site at 340 nanometers wavelength and observes the fluor-
escence with a detector responsive to the 460 nanometers wave-
length 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 from the fibrous sheet itself. The latter radi-
ation is essentially time invariant, whereas the fluorescence
from the NADPH increases with the increase in NADPH production.
Also, care is taken to avoid compressive contact of the fluoro-
meter end window with the fibrous material and the reaction
mixture to avoid variations that otherwise arise in the detected
fluorescence.
FIGURE 9a shows the variation of observed fluorescence
signal above the background fluorescence of the 903-C paper as
a function of time after the deposition of the reagent solution.
Several different glucose standard reagent solutions were used
as samples. These were prepared by dilution of a 150 Mg/Dl *
stock solution, diluted 1/30 with deionized water to yield samples
with concentrations of 5 Mg/Dl, 2.5 Mg/Dl, and 1.25 Mg/Dl, as
noted on the Figure. The sensitivity of the resultant measure-
ments is sufficiently high so that human sera which lie within
the normal range of 65-110 Mg/Dl, would require dilution of 1/30
to fall in the operating range of this chemistry.
The curves depict fluorescence that increases rapidly
through an approximately linear portion which ends at about 45
* It should be noted that as used herein, "Dl" means one-tenth
liter, and "Mg" means milligrams.
- 36 -
109~9386
seconds. The initial slopes are proportional to concentration.
The observed slope for water as sample, i.e. 0 Mg/Dl, shows a
significant variation in the first minute which results from a
variation of the optical coupling between the backing plate,
fibrous medium and fluorometer window.
A set of seventeen assays was done at the four levels
shown in FI~URE 9a. The observed initial slopes gave a linear
fit to concentration with a correlation coefficient of 0.962.
Four of these measurements were made at the 5 Mg/D1 level, and
gave a standard deviation of 0.5 Mg/Dl. By way of comparison,
the standard deviation of the background fluorescence of the
dry paper prior to administration of sample and reagents when
converted to clinical units via the fitted linear regression line
was 1.0 Mg/Dl or approximately double that observed for the rate
reaction measurement.
EXAMPTF lb
; The reagent system of Example 1 was reformulated to
minimize differential spreading and wash-out as discussed herein-
above to optimize it for the maximum linear period possible. It
was prepared from the following basic ingredients in deionized
water in the following proportions.
Concentration Commercial
Constituent (Quantity/ml) Designation
pH 7.5 Buffer
Tris (hydroxymethyl) 0.38 m (milli) Sigma Trizma T4003
aminomethane hydrochloride moles
Succinic Acid 0.12 m moles JT Baker #0346
Magnesium Chloride 0.16 m moles Sigma M-0250
Glucose-6-phosphate 20 IU (Inter- Sigma Bakers Yeast
national Units)
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1049386
1 Constituent Concentration Commercial
(Quantity/ml) Designation
dehydrogenase Sulfate Free Type
(G-6-PDH) XU # G-6378
Hexokinase 25 IU (Interna- Sigma Bakers Yeast
tional Units) Sulfate Free Type
F-300 #H-4502
Adenosin Triphosphate .02 m moles Sigma #A-3127
(ATP)
10 Nicotinamide-adenine .02 m moles PL Biochemicals
dinucleotide- #900
phosphate (NADP)
To prepare the reaction sites, twenty microliters of this reagent
solution are deposited on dry unbounded type 903-C paper, and
the paper then dried in a vacuum desiccator. The reagent solu-
tion and dried strips are stable for at least one month when
frozen.
To perform a measurement,an unused reaction site at
room temperature is placed over the window of the fluorometer of
FIGURE 6, and twenty microliters of solution are deposited as
above on the center of the treated zone. A layer of saran wrap
or other thin, optically transparent, plastic film may be placed
on both sides of the paper to eliminate direct contact of the
liquid with the fluorometer window and backing plate. This
prevents carryover of specimen from one assay to the next, but
does not affect the optical measurements. The film also maintains
wetness at the site constant by preventing evaporation. The
resulting rate reaction curves are shown in FIGURE 9b for three
concentrations and for water. They are most linear in the one
to two minute time frame, and useable at least out to three
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10~93~6
1 minutes. The observed response with water as the sample shows
similar behavior to Example la, but the measurement can now be
made after the physical situation has stabilized at one minute.
EXAMPLE lc
A slightly different result is obtained by pretreating
the type 903-C paper with a high molecular weight polymer to
minimize the absorption of solution into the paper fibers them-
selves. As one example, inch wide by eight-inch long strips are
soaked in an aqueous solution of 6 Mg/ml of polyox resin, grade
WSR-205, (molecular wt. 600,000), which is a high molecular
weight, crystalline, ethylene oxide polymer obtained from the
Union Carbide Corporation. That company advises that the follow-
ing issued U.S. patents contain disclosure of this resin and its
manufacture:
2,866,761 Hill et al - 3,062,755 Hill et al
2,897,178 Hill et al 3,085,071 Bailey
2,991,229 Ivison 3,214,387 Hill et al
3,030,315 Bailey 3,274,129 Bailey
The soaked strips are then dried in a vacuum desiccator. The
dry polyox paper is slightly thicker than untreated type 903-C
paper, measuring 0.019 inches in thickness rather than 0.017
inches.
The treated paper shows significantly reduced spreading
of liquid placea on it, which is considered desirable. The
- presumed reason for this advantage is that the treated fibers
do not absorb fluid and carry it outward. When 10, 15 and 20
microliters of liquid are applied to different sites, the
diameters of the resulting translucent spots are .375, .450 and
.475 inches respectively for untreated paper, but only .325,
.375 and .400 inches for treated paper.
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10493g6
1 Thus, the polymer resin reduces capillary spreadiny
of the fluid. Moreover, the liquid-carrying capacity of a
punched disc of the treated paper appears to be approximately
10-15 percent higher than with untreated paper. A 0.375
diameter disc of untreated paper holds roughly 38-40 microliters
of liquid without spilling over, while the treated paper will
hold 42-45 microliters.
The reaction sites on the polyox treated 903-C paper
are prepared as in Example lb. However, since the paper holds
more fluid, 30 microliters of the optimized reagent are deposited
on the desired spots and subsequently vacuum dried.
The reaction is again run on the fluorometer of FIGURE
6 by centering the dry spot over the optical window, again using
a thin plastic film to prevent carryover and evaporation, and
depositing 20 microliters of glucose solution as before.
The results are very similar to those of FIGURE 9b,
except with the time scale doubled, i.e. to six minutes. The
curve for water becomes flat at two minutes, and the slope of
the higher curves is approximately double that of FIGURE 9b,
thereby indicating roughly twice the chemical sensitivity. The
observed slope with water in the first two minutes resulted
from a fogging of the saran wrap (or backing plate itself if
saran wrap is not used) which blocks the optical coupling to the
backing plate. This was caused by a temperature differential of
roughly 5C between the backing plate and the window when the
data were taken. The slope of the blank was greatly reduced when
the backing plate was heated to a temperature closer to that of
the window. The standard curve is linear to approximately 60
Mg/Dl (1/30 dilution) so that patient sera would have to be
diluted by a factor of 50-100 to ensure that the normal range of
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104~386
1 65-100 Mg/Dl would lie within the linear range of measurement.
Note that this measurement then would consume in the order of
0.2 to 0.~ microliters of the original patient serum, and produce
the result in two to four minutes.
When the volume of glucose solution added to the dry
reagent spot is varied from 15 to 30 microliters, the observed
rates in the linear portion of the curves are found to vary only
by a factor of approximately 1.5, rather than 2 as would be
expected. (It is expected that this variation in slope can be
reduced even further when the water blank is reduced by the
proper temperature control.) This decrease in sensitivity to
the liquid volumes facilitates practice of the invention with
precise results even without corresponding precision in the
dispensing of the reactant solutions.
EXAMPLE ld
The polyox treated paper and the optimized reagent can
be used in conjunction with a press, constructed as described
above with reference to U.S. Patent 3,844,717, to assay for
glucose a whole blood sample collected on type 903-C paper. To
do this, the reaction site is prepared by preforming wells in
the treated paper by closing the press on the paper. A ring
approximately 1 mm wide and 1 cm in diameter is very highly
compressed with the center only gently compressed. Eleven micro-
liters of the optimized glucose reagent are then deposited on
the center of this well and vacuum dried. This volume reagent
just fills the center of the well and does not enter the highly
compressed ring.
To perform the measurement, a reagent-containing well
in the dry paper is centered beneath the press and covered first
with an ultrafiltration membrane and then with the paper sheet
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1 containing the dried blood spot. The blood spot is then re-
constituted with 20 microliters of saline or BRIJ*Water solu-
tion and the press closed for a period of twenty to fourty
seconds.
During this time a small volume of ultrafiltrate
containing one to ten percent of the glucose present in the
blood spot passes through thQ membrane into the well. The
volume of liquid is sufficiently small that molecular mobilities
are low and the reaction proceeds slowly, if at all. When the
press is opened and the top sheets separated, the spot dries.
The reaction site is then placed over the fluorometer window,
e.g. constructed per FIGURE 6, and 15 microliters of water added
to reconstitute the dry spot and initiate the reaction. This
volume is again chosen so that the liquid does not enter the
highly compressed ring.
The internal pressure applied by the press and the
time in which it remains closed are adjusted to bring the trans-
ferred concentration of glucose into the operating range of the
reaction.
EXAMPLE le
To further investigate the optical phenomena taking
place within the fibrous medium, simultaneous measurements of
UV transmission and reflected fluorescence were undertaken using
the above-described polyox treatea paper and optimized glucose
reagent. Two optical heads were used in the configuration shown
in FIGURE 7 with the lamp and reference detector of the upper
unit (i.e. in passages 108a and 108b, respectively) used to
provide and monitor the incident 340 nm light. The detector in
the lower unit (i.e. in passage 96b) was placed behind a 340 nm
filter while the upper detector was placed behind a non-fluor-
escing filter set centered on 460 nm. To facilitate the measure-
- ment, 3/8 inch diameter discs of polyox treated paper were
* Trade Mark
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,~...~
.. .. . .
. . . . ..
1~D49386
1 wetted with 11 microliters of the reagent solution and vacuum
dried. These were placed in a 3S8 inch diameter hole in a .015
inch thick stainless steel shim (i.e. the FIGURE 7 carrier sheet
102) which was held against the window (i.e. element 104) of the
upper instrument by the rubber gasket (i.e. element 100) of the
lower instrument. The spacing was such that the wet paper disc
under analysis was in contact with the upper window, extended
downward through the shim stock but did not contact the lower
window (element 48). The relief space (element 106) was sealed
by the gasket so that the volume in which the fluid could eva-
porate was limited. ThiS air gap also acted as a thermal buffer
to the cooler lower window.
To perform the analysis, 25 microliters of dilution
of a commercial serum standard (Ortho Diagnostics Instruments
- - ~ ~9C~f ~ ~ ~
low AOUCIIEM~ calibration fluid, lot #T~ with a glucose concen-
tration of 50 Mg/Dl were added to each of several different discs
and the resulting curves recorded. Twenty-four separate assays
were done at six different dilutions. Eight of these were at a
1/20 dilution and five used water as sample. The results for
these two concentrations are shown in FIGURE 10 along with a
scan of the paper when dry, and when wet, for comparison of
chemically induced changes with the native fluorescence and lower
transmission of the paper.
A linear regression analysis was performed on the
averaged slopes observed in each determination versus dilution.
The results show that the assay is linear from water through a
1/10 dilution. The correlation coefficient is 0.998 for the
; fluorescence measurement and 0.995 for the absorbance measurement.
The eight results at 1/20 dilution produce a standard deviation
3 in observed rate corresponding to 2.4 Mg/Dl or 4.9 percent of the
* Trade Mark
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1049386
1 50 Mg/Dl contained in the original fluid. The transmi5sion
measurments are almost as precise, showing a standard deviation
in clinical units of 5.5 Mg/Dl or 11 percent of the 50 Mg/Dl in
the original solution.
These results demonstrate that transmission measurements
can be used as well as fluorescence, provided the fluctuations
in attenuance of the fibrous medium from spot to spot are elimi-
nated. These fluctuations are apparent in the differences between
the starting fluorescence at t=O. The standard deviation of the
eight initial values in the 1/20 data set is 17.9 Mg/Dl when
converted to clinical units. The corresponding value for the
five water assays is 16.8 Mg/Dl. ThuS, the fluctuations in the
starting point far exceed the change produced by the chemical
reaction in one minute. These starting-point fluctuations are
typical, as shown by the data in the right one-third portion of
FIGURE 10, which shows the observed fluorescence and transmitted
signals when a 1 inch x 8 inch long strip of paper wrapped in
Saran wrap is pulled through the space between the upper and
lower instrument windows 98, 10 4 in FIGURE 7, with the carrier
sheet 102 removed. The fifty percent wet strip was prepared by
completely soaking one strip in water, and then contacting it
with a dry one, allowing time for the solutions to spread equally
between them. Note that the upper housing 108 of the FIGURE 7
- instrument is hingedly mounted, much like the hinged mounting
of the backing plate 82 of FIGURE 6, to facilitate access.
However, the housing 108 preferably is constrained from closing
onto the lower structure by a minimal gap, as FIGURE 7 shows.
EXAMPLE lf
Fresh whole blood is tested for its concentration of
Galactose using previously prepared reagent sites on type 903-C
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10493~;
1 paper treated with a 6 milligrams/milliliters solution of polyox
as above.
The reaction used is the enzymatic conversion of
galactose to galactonic acid by galactose dehydrogenase, with
the simultaneous conversion of Nicotinamide-adenine dinucleotide
(NAD) to its reduced form NADH.
The galactose dehydrogenase (GDH) is obtained from the
Boehringer Mannheim Corporation as a suspension of 5 milligrams/
milliliters in ammonium sulfate solution. The enzyme must be
separated from the ammonium sulfate, which is done in an ultra-
filtration cone obtained from the Amicon Corporation (Minicon 25).
One hundred twenty microliters of the GDH suspension is placed in
the ultrafiltration cone, and saline is added up to the 750
- microliter mark.
The solution is then concentrated five times to the
150 microliter mark. This wash process is repeated two more
times to yield a final solution concentrated ten times to 75
microliters. This process leaves the GDH in the cone, with the
ammonium sulfate having passed through to waste.
The GDH in the cone is removed by adding Tris Buffer
pH 6.2, .10 M up to the 750 microliter mark, and pouring the
solution from the cone into a container. The GDH concentration
in the solution is approximately 5 I.U./ml. The reagent is
completed by the addition of 6 mg of NAD.
The reagent strips are prepared by adding 15 microliters
of this solution to preformed wells in the polyox treated paper
as in Example ld, followed by vacuum drying.
It should be noted that a weak pH 6.2 buffer is used
to displace the reaction off its maximum rate which occurs at
pH 8. This is done to minimize the amount of the reaction which
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1049386
occurs during or after the ultrafiltration in the press, and
before th~ reaction is restarted on the fluorometer.
To per~orm the galactose assay, the pre-treated wells
are centered under the press, and covered with the ultrafiltration
membrane and a sheet of untreated 903-C paper. A 20 microliter
whole blood specimen is deposited on this top sheet, and the
press closed fox 75 to 100 seconds in order to achieve as high
a transfer of galactose as possible. Saran wrap is also used in
the press operation to eliminate sample contact with the press
itself and its platen.
When the press is opened, the upper layers are stripped
off and the reaction zone placed over the window of the fluoro-
meter of FIGURE 6. The reactants are reconstituted by the
deposition of 11 microliters of a 0.2 M pH 8.6 Tris Buffer, and
the reaction monitored for several minutes.
The assay is calibrated by repeating the process with
20 microliters of various standard solutions substituted for
the whole blood. The assay may also be run directly using
centrifuged and separated serum, by depositing 10 microliters
of diluted serum and 5 microliters of the pH 8.6 buffer on an
unused reagent well.
A calibration curve was obtained by assaying different
galactose standard solutions in place of whole blood. Eleven
assays were performed on nine different solutions ranging in
concentration from 0.0 to 90 Mg/DlO Linear regression analysis
yielded a correlation coefficient of 0.999, and showed that
th~ chemistry in linear up to 90 Mg/Dl. The precision of the
assay may be estimated from the difference of each of the
measured points from the fitted line. The standard deviation
of this difference is 1.48 Mg/Dl.
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104~3~6
1 A standard recovery experiment was performed by adding
galactose to four patient sera at several incremental values.
The average recovery of 10 such additions to the four sera was
96 percent, with a standard deviation of 8 percent.
The foregoing glucose analysis has been performéd
successfully with a variety of fibrous sheets, including S&S
404, 5~5 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 pre-prepared fibrous vessel, as of S~S 903-C paper with a wax
ring about the test site. The vessel is prepared by first
depositing, at the center of the test site, LDH in an ammonia
suspension, such as Sigma Type lII-Beef Heart. This is followed,
without drying, by deposition at the same point of ten lambda of
a water solution of fifteen milligrams of pyridine nucleotide
(NAD) per milliliter with a trace of surfactant such as Brij.
- The sheet is then dried in a vacuum desiccator.
;~ Where fresh serum is to be tested, the pre-prepared,
but dried fibrous vessel is first treated by depositing ten
lambda of glycine-hydrazine buffer at a pH of nine. This is ~
followed, without drying, by the deposition of ten lambda of -
a ten-to-one dilution of the serum sample. The rate of NADH
production as measured with a fluorometer in the manner of
Example 1 identifies the lactate concentration within five
percent of the amount determined by a reference procedure.
It has been found to be important in the preparation
: of the fibrous vessel that the LDH solution be added prior to
the NAD solution, otherwise the lactate analysis occurs with
significantly lesser sensitivity. It is believed that this is
. - .
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1049386
1 because the relatively heavy LDH molecules establish a bond
with the fibers or otherwise resist spreading upon the
subsequent addition of the lishter NAD molecules so that the
two reagents largely occupy the same portion of the fibrous
sheet. When the reagents are deposited in reverse order, it is
believed that the lighter NAD molecules are moved laterally
outward from the point of deposition by the heavier LDH molecules
with the result that the spot on the sheet has a concentration o~
LDH molecules within a predominantly annular concentration of
NAD molecules. The sensitivity is also observed to be diminished
when the sequence of adding buffer and serum is reversed, i.e.
when the sample serum is added before the buffer. (Note that
the addition last of sample serum for the lactate analysis of
this example is opposite to the preferred sequence for the glucose
analysis of Example 1.)
EXAMPLE III
As a third example, a fibrous vessel is again prepared
in the manner of Example II, but the serum to be tested is avail-
able 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 dry reagent sheet with an intervening
ultrafiltrate membrane, as described in the above-noted U.S. Patent
~,844,717 and illustrated herein in FIGURE 4, and placed in the
FIGURE 4 press 32 also described in full in that application.
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 BRIJ*water or saline (0.9 percent normal NaCl solution).
The press is then closed to transfer the serum ultrafiltrate
through the intervening filter sheet to the reagent sheet.
* Trade Mark
- 48 -
Jl ~
. ~
11049386
1 Alternatively, where the blood stain is not fresh, it is pre-
ferably reconstituted by the addition of twanty lambda of water
with a trace of gRI~*or like surfactant, and then subjected to
pressure to transfer it to the reagent sheet. Where the stain
is old, it has been found desirable that an initial deposition
of twenty-five to thirty lambda of diethyl ether to penetrate
the clot immediately precede the Brij water.
In either case, the press is left closed for approxi-
mately one minute. The press transfers a known portion of the
lactate in the sample to the reagent sheet, but typically in-
sufficient 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 tpH of nine) is applied
to wet the lowermost reaction sheet. Again, the rate of NADH
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 fibrous vessel.
The sample and reactant volumes set forth herein do
not saturate a reaction site having this area.
~.
; EXAMPLE IV
.~ _
As an illustration of the application of the invention
; .
to a more complex chemical reaction for blood analysis, a fibrous
vessel 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
* Trade Mark
_ 49 _
.~ ,. . ~ -.
~..~';
: '' : ':
1049386
1 example in the above-noted brochure of Boehringer Mannheim
Corporation under the heading "Neutral Fat (Triglycerides)
and Glycerol". Elowever, lipase is used to hydrolize the
neutral fat to glycerol.
The first step in the preparation of the fibrous
vessel is to apply to the center of a reaction site on S~S
903-C paper twenty lambda of lipase tSchwartz-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 gRIJ*water solution of at least three
milligrams per milliliter is deposited.
It should be noted that the three reagents so far
i added to the fibrous sheet have been added in the order of
decreasing molecular weight, i.e. the solution containing the
lowest molecular-weight glycerophosphate dehydrogenase is added
last. The remaining Preparation of the 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 lamb~a of a glycine-hydrazine buffer of pH 9
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 dry fibrous reaction sheet is now ready for
, .
* Trade Mark
-- 50 --
10~93~6
1 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 followed by
ten lambda of the blood serum being tested. The reaction site
is then examined with a fluorometer for the rate of NADH pro-
duction. A second test is performed with the same pre-prepared
reaction sheet, but at a different spot or site, 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 difference between the two rates of
NADH production, each rate being relative to the background of
the same site where that rate is measured, 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 formin~
the reaction and analaysis vessel, and hence with pure materials
the second, blank test can be eliminated.
The triglyceride concentration measured in the fore-
going manner is within five percent of the concentration as
determined with laboratory standard procedures.
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 objects 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 and in the constrution set forth
- 51 -
1049386
1 without departing from the scope of the invention, it is intended
that all matter contained in the above description shall be
interpreted as illustrative and not in a limiting sense.
It is also to be understood that the following claims
are intended to cover all of the generic and specific features
of the invention herein described, and all statements of the
scope of the invention which, as a matter of language, might be
said to fall therebetween.
'
~ 30
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