Note: Descriptions are shown in the official language in which they were submitted.
1 BACKGRO _
This application is a division oE copendiny Canadian
patent application serial number 313,670 filed October 18, 197~.
This invention,generally relates to a che~ical spot
test analysis medium,
A general object of this invention is to provide
improved compositions for chemical spot test analysis.
In one of its aspects, the inven-tion provides a chemical
spot test analysis medium of porous or fibrous character which
iO is pre-conditioned ready for receiving liquids which upon
introduction into the medium interact chemically, the medium
being pre-conditioned by an ayent which retards spreading of
liquid through the medium and which enhances the capacity of
the medium to hold liquid, the agent having hydrophobic and
hydrophilic properties.
Other ob~ects of the invention will in part appear
from the followin~ description.
~, Acaording to the invention, chemical spot test analysis '
with an optically thin concentration o~ measured reaction
product is carriecl out with a fibrous or other porous medium
pretreated with agent that conditions the medium for enhanced
containment of liquld reactants. The conditioning agent thus
increases the degree to which liquid reactants wet the medium
at a spot-like reaction site. The pretreatment of the medium
preferably also introduces at least some rea~ents. Thus, in
a typical instance, a pretreated medium which appears simply
as a dry strip of paper is put to use simply by addition o~ the
sample to be analyzed with sufficient liquid to fluidize the
. ' ', .
~)
1 reactants at a selected site~ The pretreatment of the medium
with reagents and with conditioning agent generally is confined
to the r~action sites for reasons of economy and convenience.
The conditioning agent increases the liquid-holding
capacity of the medium to contain reactants at the site in a
liquid state without undue spreading. The agent thus conditions
the medium to have a liquid absorption characteristic such
that liquid reac~ants come to rest at a higher fluid content
per unit area of the medium, but below the saturation level,
than would occur without such agents. The attainment of this
high degree of wetness at the reaction site contains a given
liquid volume in a smaller spot than would otherwise occur, and
it enhances the precision of the analytical measurement.
Useful conditioning agents appear to have a balance of
hydrophobic and hydrophilic molecules, either individually or as ``
part of a pol~mer. The hydrophobic portion presumably serves to
repel fluid from the particles or fibers of the medium, and the
hydrophilic portion presumably serves to facilitate entry of
liquid into the intexstices of the mediumO A typical
2U conditio~ing agent includes a thi~kening agent and a surfactant.
Suitable thickening conditioning agents are polyox (as set forth
; in the referenced Patent ~o. 4,059,4053,albumin, gum arahic,
guar gum~ and mannitol~ Suitable surfactants can be
selected from those available and by way of example include
Brij* The low molecular weight polyol surfactants markated
by the B~SF Corporation wlder the trade designations Pluronic*
and Tetroni~ have also been successfully used as conditioning
agents~ These are available in numerous polymeric ~orms with
varied ratios of hydrophobic and hydrophilic portions.
3~ The foregoing use of conditioning agent is considered
*Trade Mark
3.~
1 advantageous at least in part due to the finding that
measurements on reactive liquids during the production of
reaction product are preferably made with a selected range of
wetness within the structure of the reaction medium. Such
measures attain higher precision than other conditions of
wetness. The conditioning agent of this invention enhances
the attainment of the desired relatively high and stable wetness
condition during measurement. l I
In a preferred practice of the invention, the condition-
ing agent is blended in liquid form with at least some of thereactants for the prescribed analytical reaction~ ~he resultant
li~uid reagent system is applied to a fibrous sheet such as
Schleicher & Schuell type gO3-C test paper. The resultant
pretreated sheet, which after drying has a signiflcant shelf life,
is put to use for a spot test analysis, for example of blood
serum, by depositing diluted serum, and whatever further reagents
the analysis requires, on a pretreated site of the sheet. The
resultant reaction i5 monitoredt typically with a fluorometer or
other form of photometer, to measure the selected constituent-
manife6ting reaction product~
In further practice of the invention, the reactionmedium is prepared with reagents, as well as with conditioning
agent as desired, in a manner that applies differerlt reagents
within different zones of each site. This zoned introduction
of reagents is used, for example, where different reagents are
to be substantially isolated from one another until initiation of
the analysis reaction, or simply to control the reagent physical
distribution. Di~erent reagents can be zoned in accordance
with the invention by first depositing in liquid form one or
more reagents that are stable together, together with
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5s3~ `~
1 conditioning agent as suitable, to the center o~ a reaction site
and allowing the solution to spread. This is followed by
deposition at the center of the site of a selected volume of
water or other liquid to wash the previously applied reagent
outward from the center of the site. ~hereafter, and typically
after drying the medium, the further reagent is deposited at
the center of the site with sufficiently small liquid volume
so as not to spread beyond the previously-depleted central region.
After drying, the medium with reagents zoned to different
portions of each site in this manner can be stored until use
in the same manner as noted above. Where reagents are to be
zoned but without such high isolation, the wash step can be
omitted. The first deposition ofreagent, however, is generally
dried before applying the second reagentO
A sample is analyzed on a pretreated medlum by
introducing ~he sample and liquid to a reaction site, and by
.,~ . . .
monitoring the resultant constituent manifesting reaction. The
liquid required for fluidizirlg the reactants typically is
introduced with the sample as a diluent, Where the medium is a
fibrous paper-like sheet, as well as with other structures of
porous medium, it is advantageous to deliver the liquid at a
controlled rate. It has been found that too slow a delivery
of liquid tends to deplete the center of the site o reagent
constituents, whereas delivering liquid too rapidly results in
irregular and hence non-repeatable spot formatioIl. In one
preferred practice of the invention, an optimum liquid delivery
rate with a pipette forms a steady but small and stable pool of
liquid on the medium beneath the pipette tip. The optimum rate
maintains this pool, i.e. fresh liquid is delivered at
essentially the same rate at which the medium draws liquid
~ :~6~3~ ~
from the pool. Dlsapp~-arance of the pool denotes too slow a
rate of liquid delivery, and too rapid a delivery causes the
pool to become unstable so that liquid spills outward from the
pool and flows over the top of the medium.
The invention facilitates controlling the delivery
of sample liqu.id to a reaction medium site at the desired xate
by provlding a wick-like fluid delivery-controlling element
seated on the center of the site and through which the liquid
is delivered. The wick element preferably is in the form of a
1~ cylindrical disc It is significantly smaller than the spot
which the reactants form at the site, and hence covers onl.y the
central portion. For example, a cylindrical wicking disc of be-
tween l~l/2 and three.millimeter diameter has been.found.success--.
ful; in that instance ~he reactants formed a spot of roughly ten
millimeter diameter. The disc is of fibrous or other porous
structure; typically it can be punched from the same kind of
sheet usad as a reaction medium. In operation, the disc
is placed at the center of the reaction site and the diluted
sample or other li~uid is appl.ied directly to it. The li~uid
2~ enter~ the reaction medium from the disc. The wicking disc
; apparently provides a buffering capacity for excess liquid,
and appears artificially to create a liquid delivery zone
similar.to the small puddling or pool noted above as desirable
for direct application of liquid to the-medium. The disc is
readily removed from a reaction site after delivery of liquid
through it and hefore corNm~ncing measurement.- Alternatively,
the measuring instrument can accommodate the disc. ~nother
- suitable wicking element is of porous hydrophilic polyethylene.
Th~ wicking disc can ~e pretreated with reagent for
introducing the reagent to the reaction site together with the
~ 5
1 sample or other li~uid. The delivery of a reagent in this
manner is often advantageous, as where it is unstable in the
presence o~ other reagents or othe~wise is to be maintained
separate from other reactan-ts prior to commencement of the
reac-tion. The pretreatment of the disc with a reagen~ involves
absorbing a liquid solution or a suspension of the selected
reagent in the disc, and drying the disc, and where applicable
packaging it to preserve the reagent. Delivering liquid to a
reaction site through such a pretreated disc automatically
10 washes the reagent from the disc onto the site. This introduces
the disc-carried reagent to the other reagents at the desired
time and, further, with the desired controlled rate of liquid
delivery.
~ lternative to maklng measurement when reactants are
wet, the invention can be practiced, at least with some analysis
chemistries, with a measurement of reaction product when the
reactant spot is dry. Further, the invention in some instances
can be practiced, albeit with some loss of precisionj with a
single point measurement, i.e. with a single measurement of
2~ reaction product. A single measurement can be made upon
substantial completion of the constituent manifesting reaction,
or~thereafter when the reactants are dry. The basis for the
single point measurement of reaction product present in
optically thin concentration is that the signal of interest is
sufficiently distinguished from fluctuations in the background
characteristics of the medium and of the reactants. This large
manifestation o~ the reaction product relative to background
and other non-repeatable signals results from improved
chemistries ~or producing the reaction product and from improved
procedl1res which diminish the fluctuations of the background
1 slgnal. With these practices, the analysis with single-poin-t
measurement can yield precision of at least five percent~
The invention also features improvements in a
photom~tric instrument for performing spot test measurements
with optically thin concentrations of reaction product. The
instrument disposes the reaction medium in the path of optical
energy between a source and a detector by seating it between
an optical window and an opposed surface whichlis either a
second optical window or a surface of other selected optical
character, e.g. of selected reflectivity. In either event, both
surfaces which face the reaction site are closely but measurably
recessed from the medium. The intervening recess spaces
preferably are sufficient to avoid physical contact between
the medium and the surface under all normal conditions of wetness.
It has been found that this condition enhances the repeatability
and uniformity of optical coupling with the medium.
The i~ven-tion accordingly comprises the several steps
and the relation of one or more of such steps with respec~ to
each of the others, apparatus embodying features of construction,
2~ combinations and arrangements of parts adapted to efiect such
steps, and articles which possess characteristics, properties
and relations of elements~ all as exemplified in this disclosure,
and the scope of the invention is indicated in the claims.
BRIEF DESCRIPTION OF DRAWINGS
For a fuller understanding of the nature and ob~ects
of the invention, reference shGuld be made to the following
detailed description and the accompanying drawings, in which:
FIGURE 1 is a simplified showing of a fragment of
an instrument for practicing the invention;
FIGURE 2 is a side elevation view of the instrumental
3A ~
1 apparatus of Figure 1 ln use;
FIGURE 3 shows a sheet like medium and wicking disc
in accordance with features of the invention; and
FIGURES 4 throuyh 8 are graphs of fluorescence signal
showing results realized with selected practices of the invention.
DESCRIPTION OF ILLUSTR~TED EMBODIMENTS
Figure 1 shows an optical instrument 10 for performing
chemical spot test analysis and illustrating improvements which
the invention provides over the instruments which the referenced
~o 4,059,405 patent describes. The illustrated instrument has a
body 12 with a top panel 12a that receives a sheet-like fibrous
reaction medium 14 for the measurement of a reaction product at
a reaction site 14a of the sheet. The fibrous sheet 14 typically
~`~ is a strip having numerous reaction sites spaced apart from one
another and aligned in a row. The strip is recessed along one
longitudinal edge r illustrated as the back edge, with positioning
notches 16. In the illustrated arrangement there is one such
notch 16 lon~itudinally interposed between ~very two adjacent
sites 14a. An optical window 18 in the top panel of the
instrument enables optical elements within ~he instrument to
direct electromagnetic energy onto the reaction site 14a,
and enables further optical elements within the instrument housing
- to r ceive resultant electromagnetic radiation, e.g. reflected
at the wavelength of illumination or fluorescence.
Two particular features of the instrument 10 are sheet-
aligning pins 20 and 22 on the panel 12a, and a recessed surface
24 in the instrument backing head 26 that seats on the fibrous
sheet 14 above the optical window 18. The two pins 20, 22 are
selectively located on the housing panel 12a relative to the
optical window 18. The geometry is such that a reaction site
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3~ ,'
1 14a is located in optical alignment directly above the window 18
when the notches 16 are seated on the two pins 20 and 22. The
pins thus locate the fibrous sheet along both lateral axes
of the sheet to locate a reaction site in the proper measuring
position relative to the instrument window 18.
As the referenced patent sets forth, the exterior
upper surface of the optical window 18 is recessed to diminish
~le likelihood that it engages the fibrous sbeet 14. This can
be done by recessing the window below the upper surface of the
top panel 12a, or by providing a cylindrical rim 28, as
illustrated, which projects upward on the top panel beyond the
window 18. In addition, the instrument 10 has a backing head 26,
typically mounted on the instrument with a hinged or jointed
structure to allow the head to be raised away from the top panel
and alternatively be lowered to engage the fibrous sheet 14
opposite the window 18~ In accordance with the invention, the
backing head has a flat surface 24 which overlies the sheet 14
directly in register above the window 18 and is recessed away
from the sheet. In the operative position shown iIl Pigure 2.,
~0 backing the surface 24 is parallel with the window 18. A
projecting cylindrical rim 30 on the backing head extends beyond
; the surface 24 toward the housing panel 12a to engage the
fibrous sheet in opposed registra*ion with the periphery of the
window 18, i.e. in the illustrated embodiment to bear against
and engage the sheet directly opposite the cylindrical rim 28.
With this construction shown in Figures 1 and 2, the
instrument 10 positively locates a reagent sheet for repeatable,
precise measurements. The instrument lightly clamps the sheet
at the periphery o the monitored spot~like area which is in
3~ the instrument field of view. This clamping, which the
1 illustrated construction effects by the opposed rims 28 and 30
retards the loss of liquid, e.g. by evaporation, absorption or
other transfer, from the monitored area, and it securely seats
the sheet at a fixed space relative to the optical window 18
and to the backing surface 2~. The spacings of the sheet from
the recessed window 28, and from the recessed surface 24, avoid
contact of the sheet with either surface, and this has been
found to enhance the repeatability of the coupling of optical
energy between the instrument and the fibrous sheet. It will
be appreciated that the backing surface 24 can be the surface of
an optical window of the instrument 10, and generally has a
selected optical reflectance, absor~ance or other character.
Further, the internal optical system of the instrument generally
includes elements which select the field of view, which typically
ranges from between six and ten millimeters in diameter, with
eight millimeters preferred for the example set forth hereinbelow.
Alternatively, a thin opaque sheet with an aperture can be placed
over the window 18 to define the field of viaw.
By way of illustrative example, an instrument 10 as
2~ shown having an eight millimeter field of view has an optical
window of 12.5 millimeters diameter recessed by 0.25 millimeters
below the plane at which the lower surface of the sheet is
clamped (e.g. below the outer face of rim ~8), and has a flat
circular surface 24 of like di~meter and like recess from the
outer face of rim 30. ~he raised rim is typically 1-2 mm wide.
Examples set forth below illustrate chemical spot
tests analysis with an initially dry medium pretreated with
reagents so that the introduction of a diluted sample initiates
the analytical reaction. To facilitate this introduction of
liquid to the medium at a selected rate, as discussed above,
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1 the invention features the use of a small wicki~g disc 32, as
Figure 3 illus~rates. The disc is placed directly on a reaction
medium 34 at the center of the reaction site 34a at which an
analysis is to be carried out. The liquid to be delivered to
the site to initiate or carry out the analysis is applied to
the disc, rather ~,han to the medium, and the disc transfers
the liquid to the reac~ion site at the desired selected rate.
Figure 3 shows the delivery of liquid by waylof a pipette, of
which only the tip 36 is shown/ onto the top of a cylindrical
disc 32. The disc 32 controls the transfer of liquid as
described previously, and thereby facilitates the desired
delivery of liquid by relatively unskilled technicians and
without elaborate pipetting equipment,
EXAMPLE 1
Glucose End-Point Assay
A first example provides a glucose reagent: system
illustrating several features of the :invention. One feature is
use of the system for an end point assay, more particularly an
assay made with at least two measuxements at a single reacti~n
2~ site, one prior to production of the reaction product and the
other upon completion ~i.e. at the end point~ of the product~
producing reaction. The reagent system is prepared as a blend
of the following ingredients:
~
~ ~6~3~ )
1 TABLE I
GLU~OSE REAGENT
Constituent Concentration Comrnercial
(Quantity/ml) Designation
TRIS~Cl 0.563 m (milli) moles Sigma #T-3253
TRIS Base 0.437 m moles Sigma #T-1503
Succinic Acid 0.017 m moles Sigma ~S-7501
NaHCO3 0~024 m moles Baker #3506
MgC12 0.039 m moles Sigma #M-0250
Adenosine 0.033 m moles Sigma ~A-3127
Triphosphate
(ATP 3
Nicotinamide- 0.034 m moles Sigma ~N-0505
ad~nine~
dlnucleotide-
~: phosphate
(NA-DP)
Serum ~lbumin, 0~513 g~ (W/V) Slgma #A-4378
Bovine
~: Glucose-6- ~20.5 IU (International Sisma ~G-6378
Units)
phosphate
;dehydrogenase
(G-6-PD~ ~
Hexokinase 2S.6 IU (International Si~na #H-4502
Units)
Brij-35 0.03~ (W~V) Sigma #430 AG6
~ , '
- - 12
,,i
1 Features of this reagent system include relatively
high concentrations of ATP and of NADP, and the inclusion of
albumin, here in the form of bovine serum albumin, together
with a trace of surfactant, here Brij-35~ The reagent system
thus includes both reagents for carrying out the assay, and
conditioning agents for treating the medium and including both
thickening agent and surfactant. The surfactant of the
conditioning agent is understood to enhance ~he absorption by
the reaction site of the diluted sample. Without i~, the
solution appears to take longer to enter the medium, and tends
to fonm irregular spots. Reaction sites are prepared with this
reagent system by depositing twenty-five microliters of the
li~uid composition on dry, unbounded type 903-C paper. The
treated paper is dried in a dessicator to constant weight under
vacuum, and is sealed in foil packages. The resultant reagent
sites are stable for at least six months when stored frozen, and
for at least three ~onths when stored at room temperature.
To perform a glucose-assaying measurement, a fresh
reaction site is placed over the window of a fluorometer
constxucted as shown in the referenced patent with the
improvements of Figures 1 and 2. The fluorometric analysis
instrument illuminates the reaction site with excitation at
340 nm, and measures fluoresence at 460 nm. A sample of
blood serum is diluted 1:25 in water containing 1~ (V/V3 of
Brij-35 (30~ solution~. Twelve microliters of the dilution are
applied to each reaction site at a controlled rate of delivery,
for the reasons and in the manner set forth above. The
diluted sample accordingly is applied with a pipette at a
controlled rate that maintains at the reaction site center a
visible liquid pool of one to two millimeters diameter. For
\
1 this operation, the twelve microliters typically are applied in
0.5 to 1 second. Alternatively, the reagent system can be
applied using a wicking disc as described above with reference to
Figure 3~
The reaction commences substantially upon deposition of
the diluted serum, and proceeds to completion in approximately
two minutes. This is significantly faster than the glucose
reaction described in the 4,059,405 patent. IIt is understood
that the reason for this increased rate of reaction is that
the reaction site has a higher concentration of liquid, i.e. is
wetter, due to the deposition of conditioning agent a~ each
siteO A measure of the precision attained with this reaction
system and procedure is set forth in ~able II, which tabulates
measurements from six replicate samples of a mid-range blood
plasma pool having a glucose concentration of 110 milligrams
per deciliter (mg/dl). The fluorescence measured at each spot,
i.e. for each sample, initially falls from the relatively high
background value of the dry medium as the sample is added, and
has a minimum value at approximately six seconds after initiation
~0 of the reaction. It attains a maximum value, corresponding to
completion of the reaction, within two minutes.
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6~3~ ,)
1 TAsLE II
Spot_# Minimum~ R~ _ MaXimum Readinq Difference
.
1 1.05 2.77 1.72
2 1~12 2.87 1.75
3 l.ll 2.86 1.75
; 4 1.37 3.16 1~7g
1.22 3~01 1.77
; 6 1.16 2.93 1.76
______ ._______________________________________________ .. _______ ,
1~ mean 1.17 2.93 1~76
std. dev. 0.112 0.137 0.027
std. dev. 7.5 9.1 1.8
(mg/dl)
mean 110
(mg/dl)
~: ~ *
Data is expressed in relative values of fluorescence signal9
;~ Table II sets forth that,:when the difference
between the minimum and the maximum fluorescent signal
readings are used as the measure of the signal, the standard
devlation for the six replicates is 1~8 milligrams per deciliter.
By contrast, the standard deviations of the maximum readings and.
of the minimum readings, are 7.5 and 9.l mllligrams per
deciliter, respectively, which is significantly higher, Standard
~; curves;derived by plottlng the obs`erved differences as a
. function of glucose concentrations are linear from 0 to 300
milligrams per deciIiter, with correlation coefficients
typically of 0.98 or higher. Correlation studies with routine
methods for fifty-nine specimens ranging in value from 50 to
400 mg/dl of glucose showed excellent agreement, with a
correlation coefficient of 0.989. Specimens above 300 mg/dl
are re-run at higher dilution to maintain linearity.
1 It is expected that the system will yield equivalent
results at higher dilution (e.g. 1:50~ when operated on
another medium such as Whatman No. 3, which has a comparable
effective pathlength and approximately one-half the fluorescent
background as the 903-C paper. Under such improved conditions,
the linear range will be extended to 600 mg/dl.
The inclusion of conditioning agent with albumin or
other thickening agent in the glucose reagenk system of
Table I, as well as in numerous other reagent systems for spot
analysis has been found to be advantageous to stabilize the
reagent for extended storage, and to reduce the capillary
spreading of the reagent liquid on the reaction medium. It
appears that polyox, albumin and other thickening agents can be
used interchangeably, and can be provided as a mat-ter of
convenience either in the reagent system itself or as a
separate pre-reagent treatment of the reaction medium. The
important consideration appears to be that the reac,tion mixture
comes to rest within a smaller diameter on the medium with an
increased localized liquid concentration, as contrasted to
20 in~tances lacking condi~ioning agents. These chan(3es in turn
are observed to provide greater stability in the e~fective
optical pathlength, with,the net result of an enhanced measuring
precision.
It has, however, been observed that the fluorescence
measured using the foregoing glucose system exhibits a slight
increase with time, and that the rate of variation i5 similar
; both with and without glucose present in the reaction medium~
This increase is considered to result from a gradual loss of
~luid from the monitored eight millimeter diameter field of
3~ view, due to spreading of the liquid to a ten to twelve
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1 millimeter diameter extent, and due to other fluid loss
mechanisms such as evaporation. These fluid loss mechanisms
reduce the wetness of the paper, i.e. it yradually dries. The
transfer of liquid from the eight millimeter field of view to
the contiguous annular zone of the medium is considered ad-
van~ageous because it allows the reaction site to select the
volume of liquid with consequent reduced dependence on the
actual volume deposited. Another advantage is that the most
non-uniform "ringing" can be outside the field of view~
EX~MPLE II
Glucose Single Point Assay
The repea~ability of maximum readings with Example I
is limited by spot-to-spot variations in the background
fluorescence level of the 903-C paper used as the reaction
medium. Other suitable reaction media, have been found, however,
; such as Whatman paper ~3 and S&S paper #2316 and #2040B,
which have background fluorescence levels approximately one-half
that of the 903-C paper. Yet the latter fibrous media exhibit
;~ effective optical pathlengths, from multiple optical scattering
interactions with the fibers, roughly the same as for 903-C
paper. Consequently, by performing the glucose assay of
Example I on these alternate papers, one can reduce the spot-to- -
spot variations to a level below five percent of the reaction-
produced change, so that the maximum reading alone may be used~
This technique can yield a single point measurement taken after
completion of the reaction which produces the reaction product
being measured. The measurements on different samples are,
of course, made under the same condition for which the assay
is calibrated, e.g. at a specified time after commencement of
the reaction. ~s this condition of measurement is increasingly
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1 in clinical. units~ of about 1 mg/dl, which is roughly one-third
of the total. The latter precision is a fixed error independent
of glucose level. The 3.4 mg/dl error is a composite of this
fixed error and of a proportional error which adds as the
square root of the sum of squares. Thus the anticipated
proportional error is (3.42 _ 12)l/2 = 3.25 mg/dl, or 3~25~ of
the glucose concentrationO When the dilutions are increased to
lolnO, the fixed error increases to 2~, and ~hen combined with
the 3.25% proportional error still yield a composite precision of
about 4% at the 100 mg/dl level. The linear range of the assay
then extends ~o 400 mg/dl. As in Example l, the buffer zone
of the reaction medium beyond the monitored areas desensitizes
the results from variations in the volume of the di.luted sample
applied to the site~
These results demonstrate that quantitative measures
can be obtained with a reagent system which is opti.cally thin
to minimize the effects of "ringlng", and yet which is sufficiently
consentrated to produce chanyes which are large compared to the
; spot-to-spot variation of the sel~cted background.
~0 It has also been found that the same dry spots can be
measured with an instrument that responds to the reflected
; 340 nm energy. As expected for the optically thin colldition,
the reflected 340 nm signal shows a linear decrease with
increasing Glucose level, and it correlates well with the
linear increase observed in fluorescence. The precision
appears to be roughly double that observed in fluorescence.
when corrected to the lOO mg/dl level.
The utility of the foregoing technique as well as
of all the cited Examples of chemical spot test analysis in
evidence with this invention, can be extended by use of
- 18 ~
3.~'3
1 delayed beyond start of the reaction, so that the optical
pathlength at the monitored site increases, the reaction is
performed with an increasingly diluted sample in order to
maintain the optically-thin condition across the instrument
field of view.
To demonstrate the technique, the Eskala~ Glucose
tablet (marketed ~y Smith, Kline and French) was made up in
one-fifth its usual liquid volume by dissol~ing one tablet in
0.6 microliters of deionized water. Fifteen microliters of
the reagent solution prepared in this manner were app]ied to each
site on S&S ~2316 filter paper and allowed to dry. The inert
binder of the tablet appeared to function on this paper medium
similar to the thickening element o a conditioniny ayentO The
sample and the standards were diluted 1:50 in deionized water
and applied in se~uence to the sites. These were then covered
for fifteen minutes to limit evaporation while th~ reaction took
place, and the~ opened to the air to dry; this procedure is
considered to ensure that the several sites dried equally. The
absolute fluorescence levels of the monitored eight millimeter
portions of the sample spots were compared with the corresponding
levels produced on other sites by standaxd analysis solutions.
The instrument operated with the same 340 and 460 wavelengths
as in Example I, but responded to fluorescence emission trans-
mitted through the filter paper in the manner described with
reference to Figure 7 of the referenced patent.
The results showed a standard curve linear to
200 mg/dl glucose concentration~ Eight replicates of a 100 mg/dl
standard gave a standard deviation of 3.4 mg/dl. At the
same time, the measurement of the dry fluorescence of ~hirty-six
reagent-containing spots showed a standard deviation, expressed
*Trade Mark
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~6~ 3
1 other porous media with lower fluorescence backgrounds.
Further improvements can be derived using fluorescent molecules
with a higher ratio of fluorescent quantum efficiency to molar
extinction coefficien-t than NADH; examples include
Methulumbelliferone, Orthop.thaldehyde, fluorescamine, and
fluoroscein.
EXAMPLE III
The above Glucose system provides an example of a
reagent system which produces relatively large but still
substantially optically thin concentrations of NADH. The
following example illustrates an assay of blood serum for
glutamate-oxalacetate transaminase (GOT) in which the relative
changes in NADH concentration during the reaction are much
: smaller, and in which the reaction converts the fluor~scent NADH
to the non-fluorescent NAD accordi.ng to the following reacti.on
;~ Scheme
vc-Ketoglutarate + L-Aspartate- ~ L-Glutamate ~ Oxalacetate
Oxalacetate + NADH _ala e ehydrog nas~ N~D ~ L-Malate (Equation 1)
A GOT analyzing reagent system according to the
invention for deposition on reaction sites has the following
constituent concentrations.
TABLE III
Constituent Concentrati.on
... . . _ . _ _
ph 7.4 Phosphate Buf~er 0.20 moles jliter
L-Aspartate 0.144 moles/liter
~ -Ketoglutarate 0.026 moles/liter
Malate dehydrogenase 1332 IU/liter
Lactate dehydrogenase 1332 IU/liter
Polyox resin 10mg/ml
30 Brij-35 0.03~ (W/V)
1 This can be prepared using the SGOT kit available
under the trade designation stat-Pack*from Calbiochem, 10933
N.Torrey Pines Road, LaJolla, California 92037. Vial A of that
kit is reconstituted with 3.9 ml of the 1% polyox solution
containing 0.1'~ (V/V) o~ the Brij-35 solution. The resulking
reagent is four -times as concentrated as for standard usage
according to the Calbiochem directions.
Reaction sites are prepared by the deposition of twenty
microliters of this reagent system on Whatman #3 paper, with
subsequent drying to constant weight under vacuum. The mixture
contains all necessary components except the NADII, which is
added separately as described below.
EXAMPLE IIIA
The assay is run by diluting the sample o~ blood serum
1:10 in bufered diluent containing the missing NADH, and
depositing seventeen microliters of the dilution on the
previously prepared reaction siteO The decrease in fluorescence
is then monitored.
The diluent composition is:
r~ABLE IV
Constituent Concentration Source
ph 7.8 Ultra Tris Buffer 0.05 moles/liter Leon Labs lot #611015
NADH O .08 m moles/liter Calbio(hem Stat Pak,
Vial B
glycerol 1~ ~allinckrodt #5091
Lactate dehydrogenase 600 IU/liter Sigma L-1254
Brij-35 0.02~ (W/V) Sigma ~430 AG6
LDH is used in the assay to minimiæe interferences
produced by endogenous serum constitutents which vary from sample
to sample. The LDH in the diluent is prepared by adding 2.9 ml
*Trade Mark
~ 21
1 of 50~ glycerol to the Sigma vial (which contains LDH in 50~
glycerol), diluting the resulting solution 1:1000 in 50% glycerol,
and adding 0.1 ml to 4.9 ml of the NADH solution.
To stabilize fluorescence drift o~ the monitored
reaction, it has been convenient to use a partially reflecting
backing made of polished stainless steel. In the instrument of
Figures 1 and 2, this backing is the recessed surface 24~ Its
reflectance relative to a highly polished aluminum surface has
been estimated as approximately 40% by comparison of the increase
each provides in the background fluorescence level over that of
a black backing.
Figures ~A and 4B show the effect of the three backings
on the sensitivity of the fluorescence level to wetness. The
data in both Figures were rneasured on Whatman ~3 paper; in
Figure 4A the paper was untreated whereas in Figure 4B it was
treated with the polyox conditioning~agent. The. polyox data
show higher response to NADH, particularly at lower wetness~
The plotted data is normalized to have a relative fluorescence of
unity represent the background fluorescence o the initial dry
~; ~ medium. The background level of the Whatman #3 paper is the same
with and without the polyox; this level is one-half that of ~03~C
paper. The data show that the intermediate backing can give a
broad range for stable operation~ The bright aluminum backing
gives an apparent decrease in signal as the discs begin to dry
because the medium becomes less transparent, with less light
emerging to be reflected by the backing. With thinner media, a
smaller reflectance is required to achieve the desired stability.
Viewed in this context, the role of the polyox,
albumin or other thickening element in the conditioning agent
is to facilitate attaining a selected wetness in the reaction
,
- 22 -
3~3
1 site during the measuring time. The selected operat:ing range of
wetness provides maximum stability of the effective optical
pathlength and of the background fluorescence signal for the
assay. This role of the thickening element is supported by
measurements that demonstrate analyses are best performed where
results are less sensitive to wet,ness~
The desired effect is achieved in other assay systems
as well by adjusting the total solids content of the reagent
as it is applied to the reaction sites so that the diameter of
the translucent area after fluid deposition is roughly 15~
smaller (typical range 10%-20%) than or the untreated paper.
Similar decreases in spot size and/or increases in ]ocal fluid
density are observed on other media as well, such as on glass
~iber papers (Whatman GFA and GFC), microcrystalline cellulose
TLC plates (Avicel Uniplate brand available from Analtech, Inc.),
diatomaceous earth layers (pre-adsorbant layer on Quantum
Industries LQ TLC plates), and cellulose acetate electrophoresis
membranes (Instrumentation ~aboratories). By contrast, there is
; only a slight decrease in spo~ size for silica gel TLC media or
the instant TLC media available rom the Gelman Instrument Co.
under the designation I~LC SA.
Figure 5 shows the gross behavior of the fluorescence
le~ei as the SGOT reactions proceed. The changes, as graphed,
which results from the reaction are much smaller than for the
Glucose system of Example I. Note also that the 1uorescence
level falls rapidly at first as the NADH in the diluted specimen
spreads beyond the monitored area.
The fluorometer used for these measurements employs
temperature control at 30C as the referencad patent described
with reference to Figures 6 and 7. Consequently, the
:
~ 23
3~ i
1 ~luorescence also decreases as the reaction mixture equilibrates
to the 30C temperature of the window and the backlng plate.
The change in temperature from an environmental level at 25C
to 30C produces a measurable decrease in response both for the
background and for the N~DH. The temperature control maintains
30C to better than + 0.1C. This temperature dependence is
comparable in magnitude and opposite i.n direction to the ~ell
known increase in reacti.on rate of the enzyme reagent systems~
so that the two ef-fects partially compensate. However, sensible
lO error budgeting suggests that the NADH concentration (e/g/ the
total fluorescence level) be kept to a minimal l.evel so that
: its fluctuations with temperature are a small fraction of the
changes produced by the reaction.
Figure 6 plots the fluorescence signal variation of
several different levels of GOT (on an expanded scale~ and o~
several replicates at a midrange level. Usi.ng pure GOT at this
level, a coefficient of variation of five percent was observed
for seven replicates. Figure 7 presents the resu:Lts of a
comparison study with an established routine procedure for
twelve patient specimens. The correlation coefficient of the
graphed data was 0.988.
. EXaMPLE IIIB
: Figure 5 shows a relatively large initial decrease of
fluorescence signal as the diluent which carries NADH spreads
across the reaction site. It has been found that the results
for any assay (of which Glucose and GO~ are typical) are
influenced by the rate of application of the diluted specimen.
If the dilution is added too rapidly, the central area attains
a relatively higher degree of wetness which gradually equalizes
across the spot. If it is added too slowly, the solution enters
~ ~4 ~
1 tlle medium through a small central area and chromatographic
ringing is heightened~ Experience has shown that better
repeatability results when the dilution is applied a-t a rate
which maintains a slight visible pool of liquid Oll the spot
beneath the pipet tip. The pool typically has a diameter o
2-3 mm. In practice this involves deposition of 10-20 micro-
liters of solution in times of the order of one second.
As described above, it was found that sensitivity to
delivery rate could be minimized conveniently by the use of an
intermediate pad or disc punched from a thin sheet of porous
hydrophilic polyethylene or from a sheet of the 903-C or
Whatman #3 papers. The disc diameter approximates that of the
visible pool noted above; disc diameters from 1.5 ~n to at least
3 mm have been successful. The disc serves as a foc~ls for
pooling and as a reservoir to support the excess ~lui-l until
the reagent-laden site draws it off~
In operation, such wicking discs were placed on the
center of the site and the solution pipeted into the flisc, as
Figure 3 shows. After the solution was added, the disc was
removed and the reaction site examined with the fluorometer.
Results obtained weIe equivalent to those of Figure 5 and
Figure 6u The wicking discs are equally useful in other assays.
The use of NADH as a component of the diluent can be
inconvenient. As an alternative, wicking discs were dipped in
the NADH-containins diluent of Tahle IV and dried to constant
weight under vacuum. The sample was then diluted in buffer
alone, and applied to the reaction sites through these NADH-
contalning discs~ ~he concentration of NADH in the diluent
was adjusted for each type of disc to give the same increment
in fluorescence and flakness of the blank as shown in Figures
1 5 and 6~ The preferred discs were punched from a continuous
sheet of porous polyethylene (Bolab Corp. hydrophilic HDPE 35
micron pore size, l/16 inch thick, 1/8 inch in diameter) and gave
equivalent results whell dipped in the diluent of Example IIIA.
The same volume of di]uted sample was used as in Example IIIA.
Similarly good results were obtained for the Glucose
System by grindlng up an Eskalab tablet and compressing a small
amount of the resultant powder into a small tablet at the center
of a reaction site. The diluted sample was then added to the
site, eluting reagent from the compressed powder as :it: entered
the site.
EXAMPLE IIIC
. . ~ . . .~.
As another alternative to the use of MAD~I in the diluent,
a zoned reagent site was prepared bv adding the NADH to sites
previously prepared as in Exarnple IIIA. The NADH was in t:he
diluent of Table IV but prepared with ten times elevated NADH
concentration as in Example IIIA; a small volume, e.g~ three
microliters, was applied as an overspot at the center of the
previously prepared site. The sites w~re then dried for a second
-~ 20 time under vacuum. The three microliter volume was chosen to
correspond approximately to the diameter of the preferred
li~uid pool which forms as the diluted specimen is added~
.
The assay was run by depositing on the center of the
zoned site seventeen microliters of the serum sample diluted
l/10 in the diluent of Example IIIA but containing no NADH.
Figure 8 shows (with the same scale as Figure 6) the fluorescent
signal which results when samples of various concentration are
added. The sensitivity is comparable to Example IIIA, but the
results axe more variable and show steeper slopes at early times.
If larger volumes are used for the NADH overspo~, or
- 26 -
1 if the complete reagent including NADH is used in tha initial
preparation of the sites, the initial slope is still greater
and persists longer so that it is difficult to obtain a linear
portion suitable for measurement. This is understood to occur
under these conditions because the NADH forms an extremely
"ringed" spot within the instrument field of view.
The reagent systems described above and in the
referenced patent have relative concentrations o~ components
which differ significantly from their usual relationship as
1~ optimized for conventional use in liquid solution. Generally
the high molecular weight reagents, e.g. enzymes, are found at
higher concentration than for such conventional in--vitro use~
The low molecular weight, more diffusible component:s are present
at still higher concentration. Typically the enrichment ratio
for low molecular weight molecules is two to four times higher
than for the high molecular weight components~ The latter is
typically three to five times concentrated, and may range in
extreme cases as high as twenty times.
It is believed that the active enzyme rea~ents and any
other high molecular weight constituents are to be concentrated
more than for conventional practice in order to overcome an
effective diffusion barrier which results in a porous mcdium
from the unsaturated condition of the reaction site and~or
the relatively larger (vis-a-vis in vitro) surface area of the
interface of the liquid with the solution supporting surfaces.
At the same time, the diffusible, low molecular weight, components
are relatively more concentrated in order that they come to
equilibrium, after the differential chromatographic ~eparation
produced by the addition of diluted specimen, at values which
are then optimal.
~7 -
3~
The NADH concentration set forth above for the SGOT
assay is a notable excep-tion in that it has been kept low to
minimize the contribution of drifts in -temperature and wetness
on the overall assay error. For this case the xeaction kinetics
are such that the rate is not sensitive to the exact NADH
concentration, and only the upper limit of lineari-ty is
affected (e.g. the system runs linearly until the N~DE~ in the
reaction mixture is exhausted).
By way of further example, the above-noted glucose
reagent system runs to completion extremely rapidly if it is
used in a conventional liquid solution, due to the high enzyme
concentrations. It has been found that the maximum rates of
change produced in optimized enzyme assays performed on porous
media as set forth herein can approach the corresponcling maximu~
rates for the same reagents optimized for in-vitro use, when the
rate is expressed in moles of substrate converted per unit of
time and of volume, but only at the elevated concentration
described.
It is further believed that conditioning agent is
advantageous because it increases the isolation of the enzymes
from the medium structure, and of:Esets configurational c~anges
of the enzyme molecule induced by the proximity of so much surface
within the medium. Along these lines, it has been observed
that the optimized maximum rate for enzymatic reactions increases
as thP concentration of conditioning agent, and the wetness,
increase.
In summary, the improvements and features of this
invention are described above with reference to a photometric
instrument that positions a reaction site at a selected location
in a photometer field of view in highly repeatable fashion and
- 28 -
e3~
1 with operator ease. This repeatable and accurate site-locating
capabilit~, together with the selected recessing of instrument
optical surfaces from the reaction site, provide maxi~al
uniformity in the optical coupling of successively-tested sites
with the measuring optics of the instrument. I~he desirability
of an intermedia~e reflectance on the recessed surface in
attaining this objective is also set forth.
Further, the reaction sites are of a fibrous or other
porous medium that preferably is pretreated with a wetness-
enhancing conditioning agent. The agent can be app]ied inadvance of reagents, but is more conven1ently applied as part
of a reagent pre-treatment of the medium~
The practice of the invention includes delivering
liquid to a pretreated reaction site through a porous disc or
other w,icking element to control the liquid delivery to a rate-
limited but continuous application at the central portion of the
site, The wicking element facilitates attaining at the point
of liquid application a pool~like body of liquid similar to that
; which occurs naturally when li~uid is applied at the correct rate
~- wlthout a wicking e~ement. A preferred wickiny disc accordingly
has a diameter of 1.5 to 2 millimeters, although other sizes
within approximately a 1 to 3 millimeter range can be used~
Chemical analysis with the techniques set forth can
be monitored as a two-point or other rate measurement during the
reaction of interest. It can also be carried out, especially
where the monitored signal is sufficiently large relative to
nonuniform and other nonrepeatable background signals, with an
end point measurement. This measure can, in some instances,
be made after the reaction site has dried.
Chemical spot test analysis in accordance with the
- 29
1 invention employs conventional analytical reactions.
Accordingly, the constituents of reagent systems for practicing
the invention can be conventional, but as set forth above the
concentrations are significantly higher.
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 methods and in the constructions,
compositions and articles set forth without departing from the
scope of the invention, it is intended tha-t all matter contained
in the above description or shown in the accompan~ing drawings
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 generic and specific features of the
; invention herein described, and statements o-E the scope of the
invention which, as a matter of languaye, might be said to
fall therebetween.
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