U.S. patent application number 10/091296 was filed with the patent office on 2002-08-29 for element and method for performing biological assays accurately, rapidly and simply.
This patent application is currently assigned to CARDIOVASCULAR DIAGNOSTICS, INC.. Invention is credited to Oberhardt, Bruce.
Application Number | 20020119486 10/091296 |
Document ID | / |
Family ID | 21872614 |
Filed Date | 2002-08-29 |
United States Patent
Application |
20020119486 |
Kind Code |
A1 |
Oberhardt, Bruce |
August 29, 2002 |
Element and method for performing biological assays accurately,
rapidly and simply
Abstract
An element and method for easily performing liquid assays are
disclosed. The element uses capillary action to draw a
predetermined volume of a liquid sample into a reaction chamber
charged with reagent, where reaction between the liquid sample and
the reagent is monitored.
Inventors: |
Oberhardt, Bruce; (Raleigh,
NC) |
Correspondence
Address: |
OBLON SPIVAK MCCLELLAND MAIER & NEUSTADT PC
FOURTH FLOOR
1755 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Assignee: |
CARDIOVASCULAR DIAGNOSTICS,
INC.
Morrisville
NC
|
Family ID: |
21872614 |
Appl. No.: |
10/091296 |
Filed: |
March 6, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10091296 |
Mar 6, 2002 |
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09482036 |
Jan 13, 2000 |
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09482036 |
Jan 13, 2000 |
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08819341 |
Mar 18, 1997 |
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6197494 |
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08819341 |
Mar 18, 1997 |
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08247411 |
May 23, 1994 |
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5658723 |
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08247411 |
May 23, 1994 |
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07865634 |
Apr 9, 1992 |
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07865634 |
Apr 9, 1992 |
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07350851 |
May 12, 1989 |
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07350851 |
May 12, 1989 |
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07033817 |
Apr 3, 1987 |
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4849340 |
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Current U.S.
Class: |
435/6.12 ;
435/287.2 |
Current CPC
Class: |
B01F 31/00 20220101;
B01L 2300/0864 20130101; G01N 21/648 20130101; B01L 2300/069
20130101; B01L 2300/0825 20130101; G01N 2021/0346 20130101; G01N
21/51 20130101; G01N 33/525 20130101; B01L 3/502738 20130101; Y10S
436/81 20130101; B01F 23/40 20220101; Y10S 435/97 20130101; Y10S
435/81 20130101; B01F 23/56 20220101; B01L 3/50273 20130101; B01L
2400/0655 20130101; B01F 31/31 20220101; Y10S 435/975 20130101;
B01L 2400/0694 20130101; B01L 3/5023 20130101; B01L 3/502715
20130101; B01F 33/30 20220101; B01L 2300/087 20130101; B01L
2400/0633 20130101; B01L 2300/1827 20130101; B01L 2300/1861
20130101; G01N 33/4905 20130101; B01F 2101/23 20220101; B01F 21/00
20220101; Y10S 435/969 20130101; B01L 2300/0887 20130101; G01N
2201/0668 20130101; B01F 35/7172 20220101; Y10S 435/805 20130101;
B01L 3/5027 20130101; B01L 2400/0406 20130101; G01N 35/0098
20130101; G01N 2035/00435 20130101; Y10S 436/807 20130101; B01L
2300/0654 20130101 |
Class at
Publication: |
435/6 ;
435/287.2 |
International
Class: |
C12Q 001/68; C12M
001/34 |
Claims
What is claimed as new and desired to be secured by Letters Patent
of the United States is:
1. An element for performing a liquid assay, said element
comprising therein a channel structure defining a sample well and a
reaction volume in communication with each other, said channel
structure having a geometry causing a liquid sample placed in the
said sample well to be drawn into and filling the said reaction
volume via capillary action, wherein after the said reaction volume
is filled the said liquid sample remains stationary therein.
2. The element of claim 1, said element comprising a means for
monitoring a reaction in the said reaction volume.
3. The element of claim 1, said element comprising a means for
channelling light from an outside source to the said reaction
volume.
4. The element of claim 1, said element comprising a means for
detecting light emitted from the said reaction volume.
5. The element of claim 4, wherein the said means for detecting
light comprises a means for detecting scattered light emitted from
the said reaction volume.
6. The element of claim 4, wherein the said means for detecting
light comprises a means for detecting reflected light emitted from
the said reaction volume.
7. The element of claim 2, said element being disposed in close
proximity to a permanent magnet and an electromagnet, wherein the
said permanent magnet is situated between the said electromagnet
and the said element.
8. The element of claim 1, said element comprising: a base
comprising a major surface, an overlay on said base, and a cover
situated on said overlay, opposite said base; said overlay
comprising therein a channel structure defining a sample well and a
reaction space in communication with the said sample well; said
cover comprising therein a means for adding a sample to be analyzed
to the said sample well; a means for channelling light from an
outside source to the reaction chamber; and a means for detecting
light emitted from the said reaction chamber.
9. The element of claim 8, said element comprising a vent, and a
conduit in communication with both the said sample well and the
said reaction space.
10. The element of claim 1, said element comprising a liquid
absorbing matrix for withdrawing fluid from the said reaction
volume.
11. The element of claim 2, wherein the said means for monitoring a
reaction in the said reaction volume employs light and wherein at
least some of the external surfaces of the reaction slide not used
for transmission of light to the reaction volume or for detection
of light emitted from the said reaction volume are opaque to
light.
12. The element of claim 3, wherein the said means for channelling
light from an outside source to the said reaction volume comprises
an optical fiber assembly.
13. The element of claim 8, said element comprising a cover having
a length which is less than the length of the said overlay and less
than the length of the said base, said element comprising an end
cover coplanar with the said cover and spaced therefrom.
14. The element of claim 8, wherein the distal end of the said
overlay is open so that the said reaction space vents
longitudinally between the said cover and the said base.
15. The element of claim 14, said element comprising a liquid
absorbing matrix fixed to the said base and overhanging the said
distal end of the said cover.
16. The element of claim 8, wherein the distal end of the said
overlay is open so that the said reaction space vents
longitudinally between the said cover and the said base; said
overlay being provided with a first conduit communicating the said
sample well with the said reaction volume, and a second conduit
extending backward to a point beyond the sample well wherein the
end portion of the said second conduit is a means for determining
that proper filling of the said reaction volume with a fluid has
been achieved.
17. The element of claim 8, wherein the said cover comprises a
major planar segment having lateral sides bent downwardly to form
walls and then laterally to form tabs, said tabs being bound to the
said base.
18. The element of claim 1, wherein the internal surfaces of the
said element have been treated to increase their
hydrophilicity.
19. The element of claim 8, wherein the said spacer has a thickness
of from 0.001 to 0.02 inches.
20. The element of claim 16, wherein the said spacer has a
thickness of from 0.002 to 0.008 inches.
21. A method for performing an assay, comprising: adding a sample
to the sample well of an element comprising a channel structure
defining a sample well and a reaction volume in communication with
each other, wherein the said element contains a measured amount of
at least one reagent situated in the said reaction volume; wherein
a specific volume of the said biological sample is awn into the
said reaction volume by capillary anion and contacts the said
reagent to initiate a reaction between the said sample and the said
reagent, and monitoring said reaction.
22. The method of claim 21, wherein the said assay is the assay of
biological sample.
23. The method of claim 22, comprising monitoring the said reaction
by radiating light into the said reaction volume and monitoring
scattering of the light in the said reaction volume.
24. The method of claim 21, comprising: using an element containing
inert magnetic particles within the said reaction volume in
association with a permanent magnet and a electromagnet disposed in
close proximity to the said element and wherein the said permanent
magnet is situated between the said electromagnet; applying cycles
of energy to the said electromagnet to causes a change in
orientation of the said inert magnetic particles; and monitoring
the said change in orientation of the said inert magnetic particle
to monitor the said reaction.
25. An assembly Comprising at least two of the elements of claim 1
in communication with each other.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to disposable
reagent-containing elements which can be used in conjunction with
an electronic instrument for performing diagnostic assays (medical
diagnostics). It also relates to a method for performing diagnostic
biological assays employing the use of a disposable
reagent-containing element.
[0003] 2. Discussion of the Background
[0004] Many analytical techniques have been developed for chemical,
biochemical and biological assays. Procedures that use a discrete
fluid sample for the analysis of a single analyte are traditionally
characterized as wet chemical techniques or dry chemical
techniques. In recent years both types of techniques have been
automated to reduce costs and simplify procedures. Wet chemical
methods, typified by the Technicon.RTM. autoanalyzers, utilize
batches of reagent solutions, pumps and fluid controls, coupled
with conventional sensors such as radiometric (e.g.: fluorescent,
colorimetric, or nephelometric) electrochemical (e.g.:
conductometric, polarographic, or potentiometric) and others, such
as ultrasonic, etc. sensors. These techniques are characterized by
large equipment, and are generally expensive. They are complicated,
and require a skilled operator.
[0005] Decentralized testing, particularly in medical applications,
has been achieved with a variety of simpler systems often based on
cuvettes for optical determination but sometimes based on dry
chemistry-based "reagent strip" technology. Generally, a reagent
strip is an absorbent structure containing a reagent which
self-meters an applied sample and develops or changes color to
indicate the extent of reaction. Although self metering, some
reagent strips, however, require pipetting of sample to achieve
maximum accuracy and precision. A reagent strip is employed either
by itself or in conjunction with a simple instrument to read the
color intensity or hue and translate the results into a numerical
value which is displayed. Unlike a cuvette or test tube, mixing or
convection cannot be sustained in a reagent strip after the sample
has entered, and once having entered, the sample cannot be removed
from within without destroying the integrity of the strip. In one
application, reagent strip technology is used extensively in the
home by diabetic patients who test themselves daily to determine
blood sugar levels.
[0006] In a more general example of "reagent strip" technology,
Eastman Kodak has introduced a system of dry chemistry, claiming to
overcome many of the traditional weaknesses of dry chemical
methods. The Kodak technique utilizes flat, multi-layered sheets
arranged in sequence. The top layer receives a liquid sample which
passes downward undergoing separation and reactions in a
pre-arranged sequence. The sheet is designed to accept a small
volume of liquid and distribute it uniformly over a reproducible
area. The area is less than the total area of the multi-laminar
sheet. Each layer of the sheet is essentially homogenous in a
direction parallel to the surface. Once the sample has spread
radially (a rapid process), the components of the liquid can move
downward at rates that are essentially the same in any plane that
is parallel to the surface. In this way uniform reactions,
filtrations, etc. can occur.
[0007] The analyte is detected in the multi-layered sheets by
radiometric or electrochemical methods which are carried out in a
thermostated environment. This permits the use of kinetic and
static measurements to detect analyte concentrations in a liquid
sample.
[0008] Radiation is caused to enter this assembly in a path which
is traverse to the several layers. The radiation is modified by the
analyte or by a component or product of the analyte. For example,
the exciting radiation may be partially absorbed by the analyte or
by a component or product of the analyte. The modified radiation
may be reflected back transversely through the laminar assembly,
typically from a reflective layer, adjacent or nearly adjacent to a
thin layer where color is formed. Thus, reflectance can be
monitored (as opposed to transmission only through the color
producing layer). Reflectance, as expressed by Kubelka-Monk theory,
consists of optical density absorbance and scattering components
and is more sensitive than transverse colorimetry through a thin,
turbid layer. Reflectance, however, may be a more difficult
technique to standardize and to interpret data from than
colorimetry.
[0009] Conventional colorimetry has not been practiced with reagent
strips because the color producing layers are generally thin and
not transparent. The path of the exciting radiation is thus very
short (with large light losses due to opacity) and is determined by
the thickness of the layer in which the exciting radiation
encounters the substance which is excited. Since this dimension
must be very small to permit rapid measurement, e.g., 10 .mu.m to
100 .mu.m, the degree of modification of the exciting radiation is
quite small. This limits the applicability of this technique to
analyses in which the analyte (or the product of the analyte)
interacts very strongly with the exciting radiation, otherwise a
very sensitive detecting apparatus has to be used. This method has
been shown to be useful for measuring analytes in blood that exist
at relatively high concentrations, e.g., glucose, BUN, cholesterol
and albumin.
[0010] Other analytical methods have been developed that utilize
rapidly reversable chemical reactions to continuously monitor
analyte concentrations in biological fluids, or industrial effluent
streams, or ponds, lakes and streams. For example, several methods
have been proposed to measure the oxygen level in blood of
critically ill patients.
[0011] Reagent strip technology, however, possesses salient
drawbacks and limitations. For example, once a sample is added to a
reagent strip and permeates the porous structure of the strip, the
sample cannot be removed or washed out without destroying the
integrity of the strip. For example, immunoassays are extremely
difficult to perform with reagent strips, in part because
separation of free and bound antigen (or antibody) molecules from a
mixture of both cannot be readily achieved in a conventional porous
or layered structure. This limits possible immunoassay applications
to certain special cases of reactions, for example certain
homogenous reaction sequences. Incubation with mixing, a step
common to a variety of assays, cannot be performed easily in
conventional reagent strip formats since they rely on diffusion and
initial capillary action only for mixing.
[0012] Technologies have not yet been developed to cause or to
control forced convection for a specified period of time within the
porous structure of a reagent strip after the sample has entered
and permeated the porous strip structure. In conventional reagent
strips, the strip is an absorbant matrix in which mixing is
extremely difficult and limited. In addition, reagent strips almost
exclusively use reflectance as a photometric method to
quantitatively determine the extent of a color reaction. There are
no reagent strips known to the inventor which can be read via light
transmission/absorbance colorimetry, nephelometry, fluorescence,
chemiluminescence, or evanescent wave technology. Fluorescence
measurement is possible in reagent strips, but difficult to
achieve. Electrochemistry has been used successfully.
[0013] As discussed above, a large number of types of medical tests
are carried out by trained medical laboratory personnel. These
tests must be performed accurately and reproducibly with a minimum
amount of error since they are used as aids in diagnosing and
treating medical ailments. To aid laboratory personnel in
performing these tests accurately on a large number of samples in a
relatively short period of time, auxiliary equipment, which is
often expensive, is frequently used. Most of these tests are
performed on a macro scale and thus require considerable quantities
of both sample and reactants. They also require varying degrees of
sample preparation. These and other reasons are major contributors
to the generally relatively-expensive nature and inaccuracy of
medical diagnostic tests performed on body fluids.
[0014] Improvements have been made in some medical tests. For
example, the reagent strip technology discussed previously
simplifies medical tests, minimizes the required quantities of
sample and/or reactants, can minimize possible sources of error,
and lower costs. Various types of medical tests, however, have been
difficult to perform accurately and economically on either a macro
or a micro scale. In this respect, medical tests which require
rapid and thorough mixing of reagents with a sample to provide a
clearly defined starting point, an accurate measurement of reaction
time, and a clear determination of the reaction endpoint, have been
particularly difficult to perform with simple and inexpensive
devices and have been plagued with inaccuracies resulting from
errors in measurement and manipulation.
[0015] Once such type of test is the blood prothrombin time test
("PT" hereinafter). This test measures the time required to form a
blood clot (via extrinsic and common blood coagulation physiologic
pathways).
[0016] Coagulation assays, in general, are used for a variety of
reasons. They are principally used for monitoring patients
receiving anticoagulant therapy. The most frequently performed
coagulation assay is PT. Prothrombin time determinations are used
to monitor patients receiving oral anticoagulants such as warfarin.
An accurate monitoring of coagulation in these patients is
important to prevent recurrent clotting (thrombosis) and to keep
the coagulation mechanism sufficiently active to prevent internal
bleeding. Prothrombin time testing provides information to permit
better drug control to be achieved through the regulation of drug
dosage.
[0017] In conventional practice, PT assays are performed by the
addition of a liquid reagent to a plasma sample. The reagents are
typically supplied in dried form and consist primarily of
thromboplastin and calcium chloride. The dried reagent is
reconstituted before use by addition of a measured amount of
distilled water. The reagent is thermally sensitive, and
refrigeration prior to use is required. The shelf life of the
reagent in dried form is from one to two years. However, when it is
reconstituted the reagent is considerably more labile and must be
used within a few hours or discarded. In some cases reconsistuted
reagents can be kept for a few days under refrigeration.
[0018] Prothrombin time assays are performed by mixing sample and
reagent at 37.degree. C., and monitoring the progress of the
reaction until a perceptible clot (or "gel clot") is detected. The
development of a gel clot is the end point of the reaction. This
end point may be detected in various ways; by viscosity change, by
electrode reaction; and, most commonly, by photometric means. The
test result is generally compared to a result using a normal
(control) plasma.
[0019] Before performing the test, the blood sample is collected in
the tube or syringe containing anticoagulant (citrate). The blood
sample is centrifuged, and the plasma separated (e.g., by
decantation) from the red blood cells. A measured quantity (usually
0.1 ml) of plasma is pipetted into the reaction vessel or cuvet. A
measured amount of reagent is then added manually via pipette or
automatically by means of other volumetric delivery systems capable
of metering a known, preset quantity of reagent. Alternatively, the
sample can be added to the reagent directly. Typically, 0.2 ml of
reagent is employed. The addition of the reagent initiates the
reaction.
[0020] Some PT kits for use in the home are known. For example,
McCormick (U.S. Pat. No. 3,233,975) discloses a prothrombin
reaction chamber. The chamber is constructed of a transparent
material so that the progress of the reaction can be visually
monitored. To perform a blood prothrombin time test with this
chamber, one adds sequentially a measured volume of a prepared
blood sample and a measured volume of an aqueous solution of
reagent to the chamber. The chamber is then manually agitated, and
the progress of the reaction visually monitored and timed with a
stop watch.
[0021] This prothrombin reaction chamber, however, suffers from
numerous disadvantages. For the prothrombin test to be performed
with this reaction chamber, a prepared blood sample is used. Thus
sample manipulation is required. A specific volume of the prepared
blood sample must be added to the chamber. The measurements
involved in obtaining this specific volume of prepared blood sample
contribute inaccurate results and considerable labor.
[0022] This reaction chamber also requires the preparation of a
solution containing the reagent(s). The precise measurement of the
amounts of materials and water to be combined in preparing the
reagent solution introduces another additional source of error. The
measurement of the quantity of reagent solution to be added to the
chamber provides a further source of error. Moreover, as discussed
above, having to use a reagent solution is undesirable because of
potential stability problems. If the reagent solution is not used
within a few hours, the solution must be discarded.
[0023] McCormick's prothrombin reaction chamber is based on the
visual observation of the reaction to measure clotting time. It
does not permit accurate monitoring of sample mixing with the
reagent(s), accurate determination of reaction starting point
(which is as important as the end point when reaction time is being
measured), or accurate determination of reaction end point.
[0024] Accordingly there is a strongly felt need for a facile and
accurate method for the performance of biological assays, e.g., in
medical application. Such method should be based on a minimum
number of manipulations of either a sample or reagent.
[0025] Ideally it should require no sample or reagent-containing
solution preparation. It should minimize problems associated with
reagent instability and optimize accuracy. It should permit
effective mixing of sample and reagent. It should permit sample
manipulation. It should require only a very small amount of sample.
And it should be able to perform automatic treatments of the
sample, e.g., separate red blood cells from plasma in blood. This
method should be based on a simple and inexpensive
reagent-containing element.
SUMMARY OF THE INVENTION
[0026] Accordingly, it is an object of this invention to provide a
facile method for the performance of assays, e.g., biological
assays.
[0027] It is another object of this invention to provide a facile
and accurate method for the performance of assays, e.g., biological
assays.
[0028] It is another object of this invention to provide a method
for the performance of assay where the method is based on a minimum
number of manipulations of either sample or reagent.
[0029] It is another object of this invention to provide a method
for the performance of assays requiring no preparation of sample or
reagent-containing solution.
[0030] It is another object of this invention to provide a method
for the performance of assays which minimizes problems associated
with reagent instability.
[0031] It is another object of this invention to provide a method
for the performance of assays in which accuracy is optimized.
[0032] It is another object of this invention to provide a method
for the performance of assays permitting an effective mixing of
sample and reagent(s).
[0033] It is another object of this invention to provide a method
for the performance of assays permitting sample manipulation.
[0034] It is another object of this invention to provide a method
for the performance of assays requiring only a very small amount of
sample.
[0035] It is another object of this invention to provide a method
for the performance of assays permitting the automatic treatment of
the sample, e.g., separation of the red blood cells from plasma and
blood.
[0036] It is an object of this invention to provide a novel element
which permits the facile and accurate performance of diagnostic
assays.
[0037] It is another object of this invention to provide such an
element which can be used in diagnostic assays without requiring
the preparation of a reagent solution from dry reagent.
[0038] It is another object of this invention to provide such an
element permitting the accurate testing of samples with minimum
sample manipulation.
[0039] It is another object of this invention to provide a novel
element for performing a diagnostic assay in which no measurement
of sample or reagent is required for performance of the assay.
[0040] It is another object of this invention to provide a novel
element for performing a diagnostic assay permitting an
optimization of accuracy.
[0041] It is another object of this invention to provide a novel
element for performing a diagnostic assay permitting the effective
mixing of sample and reagent(s).
[0042] It is another object of this invention to provide a novel
element for performing a diagnostic assay permitting sample
manipulation.
[0043] It is another object of this invention to provide a novel
element for performing a diagnostic assay requiring only a very
small amount of sample.
[0044] It is another object of this invention to provide a novel
element for performing a diagnostic assay capable of automatically
treating the sample, e.g., separating red blood cells from plasma
and blood.
[0045] Surprisingly, all of these objects, and other objects which
will become obvious from a description of the invention provided
hereinafter, have all been satisfied with the discovery of the
present reagent-containing element for performing diagnostic
biological assays. This element comprises a channel structure
defining a sample well and a reaction volume in communication with
each other. The channel structure possesses a geometry which causes
a liquid sample placed into the sample well to be drawn into and
filling the reaction volume via capillary action, wherein after the
reaction volume is filled, the liquid sample remains
stationary.
[0046] In the assembly of this element, the element can comprise a
base, an overlay, and a cover. The base comprises a major surface.
The overlay is situated on the base. The cover is situated on the
overlay, opposite the base. The overlay comprises a channel
structure defining a sample well and a reaction space in
communication with each other. The cover comprises a means for
adding a sample to be analyzed to the sample well.
[0047] The assay is performed by monitoring a reaction of the
sample in the reaction space with the sample as a whole being
stationary during essentially all of the assay. After the assay, or
to permit manipulation of the sample, the sample can be removed
from the reaction volume, but the geometry of the present element
provides for the immobility of the sample once the reaction volume
has been filed.
[0048] Although the element can comprise the base, overlay and
cover assembly described above, it can be produced by using any
material forming technique which will produce the desired geometry.
Thus, instead of assembling the base, overlay and cover as separate
components, the element can be produced by assembling either fewer
or a greater number of components. For example, the element can be
produced by injection molding whereby one obtains two pieces which
when assembled produce the element of this invention.
[0049] In certain embodiments, the element also comprises a means
for channelling light from an outside source to the reaction
chamber. This means is referred to as a waveguide in this text.
Such an element is used with a means for detecting light emitted
from the reaction chamber. Other embodiments measure non-optical
properties of the sample. Such measurements may, for example, be
conductometric, polarographic or potentiometric in nature, or may
involve a combination of the above.
[0050] Of course, the present invention also provides a novel
method for performing a diagnostic assay. This method is based on
using the reagent-containing element of this invention. The assays
which can be performed are all liquid system assays, i.e., assays
using a liquid media. A specific example of such assays is
biological assays.
[0051] An element containing a measured amount of at least one
reagent situated in the reaction space of the element, is used. A
biological sample is added to the sample well of the
reagent-containing element. The geometry of the reagent-containing
element forces a specific volume of the biological sample to be
drawn from the sample well by capillary action to the reaction
space. In the simplest case of reagent containment, once the sample
enters the reaction space it contacts the reagent, dissolving the
reagent. Using the means for channelling light from an outside
source to the reaction chamber, light is impinged upon the reaction
space during the whole process. Light emitted from the reaction
space is monitored, permitting monitoring of the dissolution
process, the progress of the reaction, the end of the reaction, and
a determination of reaction time.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] A more complete appreciation of the invention and many of
the attendant advantages thereof will be readily obtained as the
same becomes better understood by reference to the following
detailed description when considered in connection with the
accompanying drawings, wherein like reference numerals designate
identical or corresponding parts throughout the several views.
[0053] FIG. 1 is a top view of a cover of a first embodiment of a
reaction slide according to the current invention.
[0054] FIG. 2 is a top view of an overlay of a reaction slide
according to the first embodiment.
[0055] FIG. 3 is a top view of a base of a reaction slide according
to the first embodiment.
[0056] FIG. 4 is an exploded perspective of the items shown in
FIGS. 1-3, the elements being oriented as in the assembled reaction
slide.
[0057] FIG. 5 is a top view of the elements of FIGS. 1-3, when
assembled.
[0058] FIG. 6 is an elevational longitudinal cross-section of a
first embodiment of a reaction slide according to the current
invention, the cover, overlay and base being sectioned along line
VI-VI of FIG. 5.
[0059] FIG. 7 is an elevational cross-section of a fragment of a
reaction slide according to the current invention, illustrating a
modification in which the reaction slide comprises a spacer that
includes two overlays.
[0060] FIG. 8 is a top view of a second embodiment of a reaction
slide according to the current invention.
[0061] FIG. 9 is a top view of a cover of a third embodiment of a
reaction slide according to the current invention.
[0062] FIG. 10 is a top view of an overlay of the third
embodiment.
[0063] FIG. 11 is a top view of the base of the third
embodiment.
[0064] FIG. 12 is a top view of the third embodiment.
[0065] FIG. 13 is an elevational cross-section taken along line
XIII-XIII of FIG. 12, further showing a liquid absorbing
matrix.
[0066] FIG. 14 is a top view of a fourth embodiment of a reaction
slide according to the current invention.
[0067] FIG. 15 is an exploded view of a fifth embodiment of a
reaction slide according to the current invention.
[0068] FIG. 16 is a top view of the fifth embodiment.
[0069] FIGS. 17 and 18, respectively, are elevational
cross-sections taken on lines XVII-XVII and XVIII-XVIII of FIG.
16.
[0070] FIG. 19 shows a transverse cross-sectional elevation of a
reaction slide, a preferred embodiment of a light source, also in
section, and a light detector, the light source and light detector
being disposed for making a reflectance measurement.
[0071] FIG. 20 is a top view of a reaction slide disposed in a
housing for making a reflectance measurement, a cover of the
housing being removed.
[0072] FIG. 21 is an elevational cross-section taken on line
XXI-XXI of FIG. 20, also showing the cover of the housing.
[0073] FIG. 22 is an exemplary graph showing typical results of a
measurement of prothrombin time.
[0074] FIG. 23 schematically illustrates apparatus for measuring
prothrombin time.
[0075] FIG. 24 schematically illustrates a vertical cross-section
of a modification of a reaction slide that does not employ adhesive
layers, the figure illustrating light entering that embodiment.
[0076] FIG. 25 illustrates the use of a reaction slide
(unsectioned), external waveguides and apparatus for making a
transmission/absorbance measurement.
[0077] FIG. 26 is a view similar to that of FIG. 25, illustrating
simultaneous measurements of light scattering and
transmission/absorbance- .
[0078] FIG. 27 illustrates a reaction slide and a light detector,
disposed for making a measurement based upon chemiluminescence.
[0079] FIG. 28 shows a reaction slide disposed above a partial
integrating sphere for making a measurement based on
reflectance.
[0080] FIG. 29 illustrates simultaneous measurements based on light
scattering and transmission/absorbance through the reaction space
and the use of the cover in making a fluorescent evanescent wave
measurement.
[0081] FIG. 30 illustrates the use of the base of a reaction slide
in making a fluorescent evanescent wave measurement.
[0082] FIG. 31 illustrates the use of a screen for setting up
convective currents in the reaction space.
[0083] FIG. 32 illustrates the use of a permanent magnet for
setting up convective currents in the reaction space.
[0084] FIG. 33 illustrates the use of a solenoid for setting up
convective currents within the reaction space.
[0085] FIG. 34 illustrates apparatus for producing localized
deflection of the cover to produce convective currents within the
reaction space.
[0086] FIG. 35 is a transverse elevational cross-section of a
reaction slide provided with a colorimetric transducer.
[0087] FIG. 36 is a top view of a reaction slide, with the cover
removed, the reaction slide being provided with an electrochemical
transducer.
[0088] FIG. 37 is a transverse cross-sectional elevation of a
reaction slide provided with a viscosity transducer.
[0089] FIG. 38 is a longitudinal cross-sectional elevation of a
reaction slide augmented for performing a continuous flow
measurement and having a reagent-containing layer disposed on the
base.
[0090] FIG. 39 is a fragment of FIG. 38 in the area of the reaction
space, in which the reagent-containing layer is in the form of a
reagent-containing gel.
[0091] FIG. 40 is a fragment of FIG. 38 in the area of the reaction
space in which the reagent-containing layer is in the form of a
reagent-containing membrane disposed above a liquid absorbing
matrix.
[0092] FIG. 41 is a fragment of FIG. 38, modified by the addition
of a recess in the base to accomodate a liquid absorbing matrix and
a second reagent-containing layer.
[0093] FIG. 42 illustrates a reaction slide modified for use in
initiating an assay.
[0094] FIGS. 43-51 are longitudinal cross-sectional elevations of a
reaction slide during various stages of an ELISA type
immunoassay.
[0095] FIGS. 52-60 schematically illustrate the physiochemical
conditions within the reaction space during each of the stages
illustrated in FIGS. 43-51.
[0096] FIG. 61 is a longitudinal cross-sectional elevation of a
reaction slide in which a reagent-containing matrix fills a
substantial portion of the reaction space.
[0097] FIG. 62 is a top view, with the cover partially removed, of
independent reaction spaces disposed on a common base.
[0098] FIG. 63 is a top view of a reaction slide of the parallel
flow type having three reaction spaces.
[0099] FIG. 64 is a top view of a reaction slide of the serial flow
type having three reaction spaces.
[0100] FIG. 65 is a top view of a reaction slide of the parallel
flow type having two reaction spaces, also showing light sources
and detectors.
[0101] FIG. 66 is a transverse elevational cross-section taken on
line LXVI-LXVI of FIG. 65. Together, FIGS. 65 and 66 illustrate
apparatus that may be used in conducting a Plasminogen Activator
assay.
[0102] FIG. 67 is a longitudinal cross-sectional elevation of a
reaction slide having a vent cover useful in selective venting.
[0103] FIG. 68 is a top view of a reaction slide provided with a
pinch valve.
[0104] FIG. 69 is a section taken on line LXIX-LXIX of FIG. 68,
also showing a push rod for actuating the pinch valve.
[0105] FIG. 70 is a top view of a reaction slide having an
auxiliary conduit.
[0106] FIG. 71 is a schematic representation illustrating how two
liquids may be selectively introduced into a common chamber.
[0107] FIGS. 72-74 schematically illustrate various forms of
cascading.
[0108] FIG. 75 is a longitudinal vertical cross-section of a
reaction slide together with apparatus for using suspended magnetic
particles to measure a coagulation reaction.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0109] Shown in FIG. 1 is a top view of a cover 10 of a first
embodiment of a reaction slide according to the current invention.
Shown in FIG. 2 is a top view of an overlay 20 of the first
embodiment. Shown in FIG. 3 is a top view of a base 30 of the first
embodiment.
[0110] FIG. 4 is an exploded view showing the relative positions of
the cover 10, overlay 20 and base 30.
[0111] FIG. 5 is a top view of the cover 10, overlay 20 and base
30, when assembled.
[0112] FIG. 6 is a longitudinal vertical cross-section of a first
embodiment of a reaction slide 1 according to the current
invention. The cover 10, overlay 20 and base 30 are sectioned along
line VI-VI of FIG. 5. As will be described more fully below, the
reaction slide 1 contains certain elements in addition to those
shown in FIGS. 1-5.
[0113] Now referring generally to FIGS. 1-6, the cover 10 comprises
a thin glass or polymeric sheet, typically transparent, having
formed therein a sample receiving opening 14 and an elongate
opening 12 proximate a distal end 16 of the cover.
[0114] The overlay 20 comprises a thin glass or polymeric sheet,
typically transparent, having formed therein a cut-out, the cut-out
having a geometry as shown to form a sample receiving opening 22, a
reaction space 24 and a conduit 26 communicating the reaction space
and the sample receiving opening. (The reaction space 24 becomes a
reaction volume upon assembly of the cover, overlay and base.)
Advantageously, tapering walls 25 form a transition between the
conduit 26 and reaction space 24. The distal end 28 of the overlay
is closed as shown at 29.
[0115] The base 30 comprises a sheet of glass or polymeric
material, typically transparent and typically somewhat thicker than
either the cover 10 or overlay 20.
[0116] The cover 10 and base 30 are separated by a spacer 60 (FIG.
6), the spacer 60 being made up of the overlay 20 sandwiched
between two adhesive layers 62 which respectively join the overlay
20 to the cover 10 and the overlay 20 to the base 30. Each of the
adhesive layers 62 has the same shape as the overlay 20. That is,
each of the adhesive layers is formed with an opening having a
shape corresponding to the sample receiving opening 22, the
reaction space 24 and the conduit 26 of the overlay 20.
Accordingly, there are formed in the reaction slide a sample well
64, a reaction volume 66, a conduit communicating the reaction
volume 66 and the sample well 64, and a vent 76 formed by the
opening 12 in the cover 10 communicating the reaction volume 66
with the environment of the slide.
[0117] The bottom surface of the cover 10, facing the base 30, is
spaced from the top surface of the base 30 by a distance that is
sufficiently small to cause a sample placed in sample well 64 to be
drawn into the reaction volume 66 by capillary action. Such action
is made possible by the presence of the vent 76.
[0118] As shown in FIG. 5, the length (left to right in the
drawing) of the cover 10 is the same as that of the overlay 20, and
the width (top to bottom in the drawing) of the cover 10 and
overlay 20 are the same and are less than that of the base 30.
[0119] Preferably there is provided a liquid absorbing matrix (LAM)
for withdrawing fluid from the reaction space when desired. To this
end, there may be provided as shown in FIG. 6 a LAM assembly 50
including a LAM pad 51, illustrated as a sponge, fixed on a LAM
support 52, the LAM support 52 comprising an arm 53, a tab 54 fixed
on the base 30, and a living hinge 55 joining the arm 53 and tab
54. When the arm 53 is pressed downwardly, manually or by an
automated presser (not shown), the LAM 51 will enter the vent 12
and make contact with fluid in the reaction volume 66, thereby
drawing out the fluid. It has been found that, when the cover 10 is
made of a polymeric material, this withdrawing action may be
enhanced by downward deflection of a portion of the cover 10
adjacent the vent 12. It would appear that this enhanced withdrawal
is caused by a localized narrowing of the distance between the
cover 10 and base 30, thereby creating a narrowed passage to
enhance capillary action. Thus although the cover 10 can be made of
either rigid of flexible material, in this embodiment or the
invention, the cover 10 is preferrably made of a flexible
material.
[0120] Observations and measurements of chemical reaction occurring
within the reaction volume 66 may be made by a number of methods,
as described more fully below. At present, optical methods are
preferred, but the choice of method will depend upon the assay
being performed. Shown in FIG. 4 are a number of paths that light
may typically follow for making such measurements and observations.
These paths may be used alone or in combination.
[0121] In light path 40, light is introduced through a side of the
overlay 20 and passes initially through a portion of the overlay
disposed between the closed end 29 and tapering wall 25. This
portion of the overlay and its opposite corresponding portion will
be referred to as internal waveguides 27. Thereafter, the light
passes through the reaction volume 66 and out through the opposite
waveguide 27. As illustrated schematically, light passing in this
direction through the waveguides is internally reflected off the
top and bottom surfaces of the overlay 20. Light path 40 is useful
in making measurements based on the transmission or absorbance of
light by the fluid within the reaction volume 66, in which case
there is measured the ratio of light intensity before and after
passing through the sample in the absence of scattering or
excluding scattering. The Beer-Lambert Law describes the
phenomenon. Standard detectors are employed in a line of sight
configuration with the light source.
[0122] Light path 41 illustrates a measurement that may be made
based upon light scattering in which light is first introduced
transversly through an internal waveguide 27, enters the reaction
volume 66, is then scattered by the sample, a portion of the
scattered light proceeding downwardly through the base 30 and then
leaving the reaction slide. Light scattering measurements or
nephelometry measures light which is not irreversibly absorbed by
the sample and emerges at various angles, the spatial/intensity
distribution being dependent upon particle size, shape and
wavelength of the excitation energy. Rayleigh and Mie theories are
useful models. Standard detectors are employed. Examples are
photocells or photomultipliers, the latter being employed at very
low light levels. Excitation source wavelength may be fixed at a
particular value. The detector is typically set at a predetermined
angle from the direction of excitation.
[0123] Light paths 42 and 43 respectively show light entering
laterally through the sides of the cover 10 and base 30,
experiencing total internal reflection as it passes directly above
and beneath the reaction volume, respectively, and exiting through
the opposite edge of the cover or base. As will be explained more
fully below, such light paths may be employed for detecting
fluorescence using an evanescent wave measurement.
[0124] Other light paths are possible, including vertical paths
passing through the cover, reaction volume and base and light paths
making use of reflectance off a sample in the reaction volume,
according to which light may both enter and leave the reaction
volume by way of the cover 10 or base 30.
[0125] It will typically be desirable to exclude stray light from
entering the reaction slide. For this purpose, any external surface
of the reaction slide which is not to be used for the transmission
of light may desirably be painted with an opaque paint. The choice
of surfaces to be so painted will be governed by the assay to be
performed and the elected methods of measurement. When any of the
components 10, 20 or 30 will not be used for the transmission of
light, that component may be made of a material which is itself
opaque, such as metal.
[0126] When using light-paths such as 40 and 41 in FIG. 4, it
becomes important to transmit as much light as possible through one
or both of the internal waveguides 27, keeping the losses as low as
possible. It has been found that the presence of the adhesive
layers 62 can cause the spacer 60 to perform like an optical fiber,
the waveguides 27 corresponding to a core of an optical fiber and
the adhesive layers 62 corresponding to cladding.
[0127] Refractive index mismatch between the waveguide 27 and the
adhesive layers 62 produces total internal reflection of light
striking the interface at angles greater than the critical angle.
By way of example, reference is made to FIG. 24, wherein there are
shown core 70, cladding 71 surrounding the core 70, and incident
light ray 72 striking and passing through the core 70. The core 70
may correspond to the internal waveguide 27 of the overlay, and the
cladding 71 may correspond to the adhesive layers 62. If, for
example, the core material 70 has a refractive index n.sub.1 of
1.62 and the cladding 71 has a refractive index n.sub.2 of 1.52,
the sine of the critical angle is n.sub.2/n.sub.1, or
1.521/1.62=0.938. The critical angle is then 69.8 degrees.
[0128] Referring now to FIG. 7, there is shown a fragmentary
vertical cross-section of a modification of the embodiment of FIGS.
1-6, the view of FIG. 7 being taken at a representative location
corresponding, for example, to the extreme left-hand portion of
FIG. 6. In this modification, the spacer 60 includes a second
overlay 68 which is substantially identical to the overlay 20. A
third adhesive layer 62 is used to join the overlay 20 and the
second overlay 68. Because the second overlay 68 is identical to
the first overlay 20, it forms a second pair of internal waveguides
27. The additional cross-sectional area provided by the additional
waveguides 27 substantially increases the amount of light that may
be introduced into the reaction volume 66 through the internal
waveguides 27.
[0129] Referring now to FIG. 8, there is shown a top view of a
second embodiment of a reaction slide 1 according to the current
invention. The base 30 and overlay 20 of this embodiment are
identical to those shown in the embodiment of FIGS. 1-6. However,
the cover 10 has a length that is less than the length of the
overlay 20. There is provided an end cover 75, coplanar with the
cover 10 and spaced therefrom to form a gap. This gap creates vent
76, communicating the reaction volume 66 with the environment of
the reaction slide 1. For the sake of clarity, the LAM assembly 50
is not shown.
[0130] Additional variations of the above-described embodiments are
possible. For example, it is not necessary for a LAM assembly to be
fixed on the base 30. Such an assembly may be provided separately
and may be manipulated manually or using an automated system.
[0131] The adhesive layers 62 may be omitted, and an alternative
method such as heat sealing may be used to join the cover 10,
overlay 20 and base 30. In such a case, the spacer 60 is formed
entirely by the overlay 20. Referring again to FIG. 24, in such a
case the internal waveguide 27 of the overlay 20 will again act as
a core 70 of an optical fiber, but the cladding 71 will be formed
by the cover 10 and base 30.
[0132] FIGS. 9-11 show top views of, respectively, a cover 10,
overlay 20 and base 30 of a third embodiment of a reaction slide
according to the current invention. FIG. 12 shows a top view of the
assembled reaction slide of this embodiment. This embodiment
differs from that of FIGS. 1-6 in the omission of conduit 26 and in
that the distal end 28 of the overlay 20 is open, so that the
reaction space vents longitudinally between the cover 10 and base
30 instead of vertically through the cover.
[0133] The cover 10, overlay 20 and base 30 may be secured to each
other using adhesive layers 62 as described in connection with the
previous embodiments, or the adhesive layers 62 may be omitted and
the various elements of the reaction slide may be joined by heat
sealing or solvent bonding, etc. . . . It has been found that,
where heat sealing is employed, discontinuities in the heat seal
often result, impairing the total internal reflection of light when
passing through the waveguides 27. To compensate for such
impairment, a reflective layer 78 may be placed atop the waveguide
27 of the overlay 20 through which light will be introduced. A
corresponding reflective layer 80 may be placed on the base 30.
Such reflecting layers also may be used in other embodiments
according to the current invention, if desired.
[0134] FIG. 13 illustrates the open-ended reaction volume of the
embodiment of FIGS. 9-12, together with the addition of a LAM 82 in
a configuration preferred for use with such an open-ended reaction
volume. In particular, the LAM 82 is fixed on the base 30 and
overhangs the distal end 16 of the cover 10. When it is desired to
remove liquid from the reaction volume 66, the LAM 82 is depressed,
resulting in a localized deformation of cover 10 and LAM 82,
causing contact between the LAM 82 and the fluid in the reaction
volume 66 for the withdrawal of the fluid.
[0135] Shown in FIG. 14 is a top view of a fourth embodiment of a
reaction slide 1 according to the current invention. In this
embodiment, the distal end 28 of the overlay is open, as is the
case with the embodiment of FIGS. 9-13, such that the reaction
volume 66 vents between the cover 10 and base 30. The cover 10 is
shorter than the overlay 20, so that a portion of the overlay 20
may be seen extending to the right in the drawing from beneath the
cover. The overlay 20 is provided with a first conduit 90
communicating the sample well 64 with the reaction volume 66 and a
second conduit 92 extending backward to a point beyond the sample
well, such that the end portion 94 of conduit 92 extends beyond the
edge of the cover. The second conduit 92 and its end portion 94 are
used for visual inspection to determine that proper filling has
been achieved.
[0136] In particular, in a typical use of a reaction slide
according to the current invention, that portion of the reaction
slide containing the reaction volume 66 will be disposed within a
measuring instrument, whereas that portion of the reaction slide
containing the sample well will extend out of the measuring
instrument so that a sample may be introduced into the sample well
64 when desired. When the sample passes from the sample well 64
into the reaction volume 66, it no longer is visible to the user,
that portion of the reaction slide being disposed within the
measuring instrument. Accordingly, when the user observes the
presence of sample in end portion 94 of second conduit 92, it is
assured that proper filling has been achieved. It may be seen that
a reaction slide according to this embodiment is most useful in
those cases when the cover 10 is opaque. In the alternative, if the
cover is transparent, substantially all of the second conduit 92
may be used for visual observation of proper filling. In such a
case, it is not necessary that the second conduit 92 extend beyond
the end of cover 10 as illustrated, and the length of the cover 10
may be the same as the length of the overlay 20.
[0137] Second conduit 92 is not essential, as proper filling may be
monitored by electro-optic means using the same light detectors
used in monitoring the results of the assay being performed.
Indeed, the embodiments of FIGS. 1-13 do not employ a second
conduit 92.
[0138] FIG. 15 is an exploded view of a fifth embodiment of a
reaction slide 1 according to the current invention. A top view of
this embodiment is shown in FIG. 16, with selected vertical
transverse cross-sections being shown in FIGS. 17 and 18.
[0139] There is shown a base 30 on which is fixed an insert 110 and
insert cover 100. Insert cover 100 is generally formed by a major
planar segment 101 having lateral sides bent downwardly outward to
form walls 104 and then laterally to form tabs 106. The tabs 106
are bonded to the base 30 with the insert 110 being disposed
between the planar segment 101 of the insert cover 100 and the base
30, the height of the walls 104 generally corresponding to the
height of the insert 110.
[0140] Insert 110 includes a sample receiving opening 112
communicating with a conduit 114 which ends in outwardly tapering
walls 116. As the length of the insert 110 is substantially less
than the length of the insert cover 100, a reaction volume 66 is
formed to the right of the insert as shown in FIG. 16.
[0141] Thus, it may be seen that the walls 104 in the area of the
reaction volume 66 serve the functions of the internal waveguides
of the previous embodiments, and for this purpose at least those
portions of the walls 104 that are disposed in the vicinity of the
reaction volume 66 are made of a transparent material.
[0142] Although the insert 110 may be bonded to the base 30,
variations are possible. For example, the insert and base may be
formed as one piece, molded or machined to the appropriate shape
and channel structure.
[0143] As will be described in more detail below, an assembled
reaction slide according to the various embodiments will typically
contain one or more reagents specifically selected for their
utility in performing any of the many assays that may be performed
using reaction slides according to the current invention. For
example, liquid reagent may be placed in the reaction volume by
filling through the sample well or, preferably, through the vent.
The reagent can then be freeze-dried, the exact conditions of the
freeze-drying process being dependent upon required optima and the
type of reagent employed. There is thus produced a reaction slide,
ready for use, having a premeasured amount of reagent disposed
therein.
[0144] Typically, it may be desired to modify the internal surfaces
of the reaction slide which will contact the sample or reagent or
both to modify the liquid/solid/air contact angle of the surfaces,
the surfaces thus being treated to increase their hydrophilic
character. Such treatment will increase the ease with which the
sample flows from the sample well to the reaction volume.
[0145] There are a variety of methods available for decreasing
contact angle on a hydrophobic (or nonpolar) surface, thereby
rendering it more hydrophilic. Surface active agents (or
surfactants) which are typically employed as wetting agents may be
used. For example, small amounts of Triton type dispersion agents,
Tween (polyoxyethylene derivatives of fatty acid partial esters of
hexitol anhydrides) type surface active agents, and Brij
(polyoxyethylene ethers of higher aliphatic alcohols) type wetting
agents may be utilized. Surface modification via chemical
derivitization of surface molecules can create polar prosthetic
groups. Other techniques include surface modification using
controlled electrical discharge or plasma treatment.
[0146] It should be noted that the height of the reaction volume is
critical and is defined by the thickness of the spacer 60. This
height should be uniform and can range from 0.001 to 0.02 inches
(approximately). Typically, this height is preferably from 0.002
inches to 0.008 inches, and most preferably approximately 0.006
inches.
[0147] This order of magnitude is not only appropriate to achieve
functional capillary action in the channels but is of the same
order of magnitude as is required, generally, for optical waveguide
transmission of light by total internal reflection. Coincidentally,
this dimension is approximately of the order of magnitude required
to produce preferential phase separation to the center of a flowing
stream of suspended particulate or cellular material in a two phase
system (or suspension) during sustained laminar flow conditions
which may be achieved, as will be described below.
[0148] For construction of the reaction slide, all materials which
come in contact with sample or reagent should be relatively inert.
The surface properties of the materials should be such that
appropriate wetting of the surface is achieved by the sample to
provide proper flow conditions. Generally a low contact angle is
best.
[0149] Cover 10 may be fabricated from a solid thin sheet of
paramagnetic material or a laminate consisting of a coated
paramagnetic material or could be fabricated from plastic or
glass.
[0150] The paramagnetic material could be iron or nickel.
Chemically inert thin coatings, such as polyvinyl chloride,
acrylic, or polycarbonate could be utilized. A polymer with
encapsulated iron oxide (e.g., magnetite) could be utilized as
well.
[0151] The cover also could be fabricated from a variety of glasses
and fused quartz. Polymeric materials which could be advantageously
utilized include: polycarbonate, PET, PETG (glycol-modified
polyethylene terephthalate), acetate, acrylonitrile, and cellulose
nitrate. A variety of coextruded films, composites and polymer
alloys may also be used. Of primary importance are dimensional
stability, stiffness, resiliency, and optical clarity (when
required). The ability of a material to be fabricated in thin
sheets is also a factor. Methyl methacrylate and polystyrene are
both potentially suitable materials but are difficult to fabricate
in thin sheets.
[0152] The cover is typically of greater surface area (or projected
area) than the reaction volume. The cover may typically assume the
same length and width as the spacer (e.g., 2 inches.times.. 0.5
inches) but could be larger, if required, or smaller.
[0153] Materials which may be utilized to produce a good to
excellent overlay include: polycarbonate, PETG, methyl
methacrylate, polystyrene and glass. However, glass is difficult to
fabricate into the required shapes. Materials which may be utilized
to produce a good to moderately good overlay include: polyvinyl
chloride, nylon (polyamides), PET or polyethylene terephthalate
(e.g. mylar), and acetate. Materials which may be utilized to
produce an acceptable overlay include: acrylonitrile, low density
polyethylene film, PP/EVA coextruded film, EVA/nylon/EVA coextruded
film, PP/EVA/PE/EVA coextruded film, and oriented polypropylene
film. Materials which may produce an overlay of marginal
acceptability include: XT and high density polyethylene film. In
general, the better materials provide better waveguides because
they have lower light scatter losses and transmit well in the
visible spectrum where the most commonly employed excitation
wavelengths may be found.
[0154] There are many adhesives which can be employed to secure the
overlay to the cover and base. Acrylic adhesives are generally
good. The best adhesives retain some flexibility, are transparent,
and have low light scatter losses when cured, pressure treated, or
otherwise activated. The length and width of the overlay may be
varied over a wide range, but could be typically and approximately
2 inches.times.0.5 inches on a 3 inch.times.0.75 inch base.
Thickness of the spacer is typically in the range of 0.002 to 0.010
inches. Thinner spacers may occasionally result in impeded
capillary channel flow. Thicker spacers tend to lose liquid at the
air interface adjacent to the edge of the reaction volume due to
poor capillary action at larger diameter conduits.
[0155] The base is a solid support and can be made from a variety
of materials. It should be rigid enough to maintain and support the
reaction volume geometry, transparent in the reaction volume region
(if required for monitoring) and capable of being bonded to the
spacer/cover component. Fluorinated hydrocarbons such as Teflon
make poor bases because they are difficult to bond. Glass is an
acceptable material. Excellent bonding may be achieved with
polycarbonate or methyl methacrylate base materials. A typical
minimum thickness for the base is approximately 0.020 inches for a
material such as polycarbhonate. An aluminum base (if transparency
is not required) could be thinner. If the base is too thin, it may
bend too easily and alter the volume of the reaction volume
unintentionally during handling or manipulation during an assay. If
the base is too thick, it may take too long to achieve thermal
equilibrium for a temperature controlled assay. This is especially
true for materials with low heat conductivities. The length and
width of the base are variable. The base could be as small as 0.25
inches in width and 1 inch in length (or even smaller). Typically,
the base will be approximately 0.75 inches in width and 3 inches in
length. This provides enough room for a sample well, connecting
conduit, and reaction volume with vented end. There would also be
an area to grip the slide with thumb and forefinger for handling
and placement and another area for an optically or magnetically
readable code to provide information to the analytical instrument
employed. This information might include the type of assay, control
parameters, calibration information for that batch of reagent, etc.
As will be described later, the base could be wider (or longer) if
multiple assays are to be performed on the same sample. In such a
case, multiple reaction spaces might be used in parallel (or
series) communication with the sample well. The base could consist
of a composite material (e.g., two layers, such as a lower layer of
aluminum, iron, or other metal with a hole under the reaction
volume. Atop this layer and affixed thereto would be an upper layer
of transparent material, such as polycarbonate, which would define
the bottom of the reaction volume to allow light transmission
through the hole in the lower layer.
[0156] When used, the reflective layer (78, 80 FIGS. 10, 11) can be
made by applying a thick film of aluminum paint. Other methods
include chemical deposition of silver metal and vacuum vapor
deposition of silver or aluminum. Another fabrication technique is
to employ metallized heat sealable film, for example, metallized
linear low density polyethylene (LDPE) film of approximately 20
microns thickness (or less). Other metallized polymer films may
also be utilized if coated with a heat sealable material such as
polyvinylidene chloride. An example is metallized heat sealable
polypropylene film (polypropylene coextruded with heat sealable
materials). Other possibilities include metallized cellophane
coated with a heat sealable material. Metallized films may be heat
sealed or glued with an adhesive (e.g. cyanoacrylates) to the base
and cover of the reaction slide. Metallized glass may also be
utilized.
[0157] As stated above, a currently preferred method of use of a
reaction slide according to the current invention involves the use
of one or more sources of light external to the reaction slide and
one or more light detectors external to the reaction slide.
Illustrative examples of such instrumentation and use will now be
described.
[0158] Shown in FIG. 19 are a transverse vertical cross-section of
a preferred embodiment of an external light source 120, a
transverse vertical cross-section of a representative embodiment of
a reaction slide 1, the cross-section being taken in the region of
the reaction volume 66, and a light detector 121 disposed beneath
the reaction slide 1 in the area of the reaction volume 66. A dried
reagent 125 is deposited on the walls of the reaction volume
66.
[0159] In this embodiment, the light source 120 comprises a plastic
housing 130 supporting an LED 132 having electric leads 134. As
shown, a step 136 is formed in housing 130. As shown, the cover 10
may be made of an opaque material such as a metal.
[0160] In use, the light source 120 and reaction slide 1 will mate
such that the step 136 receives the base 30 of the reaction slide
and the LED 132 is disposed above the base 30 and in contact with
or closely adjacent an internal waveguide 27 of the reaction
slide.
[0161] The arrangement illustrated in FIG. 19 is designed to employ
a light path such as that shown at 41 in FIG. 4. A more detailed
example of instrumentation for accomplishing such measurement is
shown in FIGS. 20 and 21. Housing 140 comprises lower housing 142
and cover 144 resting on or integral with lower housing 142. A
lower end of wall 146 of cover 144 is spaced from the top 148 of
lower housing 142 by a distance which is sufficient to allow the
reaction slide 1 to be inserted. Lateral guides 150 and stop 152
establish a proper position of reaction slide 1 for a measurement.
Light source 120 and light detector 121 are disposed within the
housing 140, as shown. There is provided in the top 148 of the
lower housing an opening 154, disposed immediately beneath the
reaction volume of the reaction slide such that light passing
through the base 30 of the reaction slide may reach the light
detector 121.
[0162] Sample well 64 of reaction slide 1 is disposed outside the
housing 140, such that the reaction slide 1 may be inserted into
the housing 140 before a reaction is initiated.
[0163] The light detector 121 may be used to monitor the progress
of sample entry into the reaction volume 66 and the subsequent
progress of the reaction within the reaction volume 66. The light
source 120, reaction volume 66 and light detector 121 are disposed
in a portion of the instrument that is isolated from ambient light.
Desirably, those portions of the reaction slide 1 which are exposed
to ambient light are made of opaque materials or are painted so as
to aid in the exclusion of stray light from the reaction volume
66.
[0164] Temperature control is provided for the reaction slide by
means of heaters of a thermal control system, illustrated
schematically as element 156. One form of such a heater may be a
conductive heater strip 157 fastened to the bottom of plate 148.
Regardless of the form of thermal control system used, it is
desirable that it be capable at least of maintaining the
temperature of the plate 148 at 37.degree. C.
[0165] The instrument shown in FIGS. 20 and 21 is not of a standard
commercially-available type but is instead custom designed for use
with reaction slides. As in the case of other instruments currently
available for decentralized testing, optical or magnetic code
reading capability is preferably present to provide for
identification of the assay to be performed and of the particular
reaction slide being used, along with calibration. information.
Such a code may be affixed to the reaction slide during
manufacture. In addition, other structure may be present to provide
for mixing when required (to be described below). As also described
below, there will desirably be associated with the illustrated
instrumentation a system microprocessor, display or other data
presentation means, any necessary analog-to-digital converters,
power supplies, and the like.
[0166] As may be seen from a consideration of FIG. 21, it is
desirable for the spacing between the lower end of wall 146 and the
plate 148 to be as low as possible to aid in the exclusion of
ambient light. Accordingly, this embodiment of instrumentation is
not conducive to the use of a reaction slide that is provided with
a LAM assembly 50 as shown in FIG. 6. Therefore, there may be
provided within the housing 140 a movable structure for advancing
and withdrawing a separate LAM from the vent 76.
[0167] One type of assay that may advantageously be conducted using
the apparatus illustrated in FIGS. 20 and 21 is a measurement of
prothrombin time. Illustrative results of such a measurement will
now be described with reference to FIG. 22.
[0168] FIG. 22 shows a resulting light scatter curve obtained when
a plasma sample is deposited in the sample well 64 of a reaction
slide containing dried thromboplastin calcium reagent. This curve
is unexpected and quite different from the 900 light scatter signal
observed for the same assay using liquid reagents. The same type of
photodetector was employed in earlier work with liquid reagents.
See for example Oberhardt, B. J., Monitoring System for
Fully-Automated Prothrombin Time Determination, Digest of the 7th
International Conference on Medical and Biological Engineering,
Stockholm, Sweden, 1967, p. 187, which is hereby incorporated
herein by reference. With liquid reagents, however, different and
considerably less informative curves result. An apparatus using the
same photodetector as in the present invention and which employed
liquid reagents to produce the previously published curves is
disclosed in U.S. Pat. No. 3,450,501. This patent is hereby
incorporated herein by reference.
[0169] In prothrombin time determinations that are performed with
liquid reagents and plasma samples, the scatter intensity curve
starts off low, increases upon addition of reagent, and increases
further at the formation of the gel clot.
[0170] The different result obtained with dried reagent may
possibly be explained as follows, although the present invention is
by no means to be construed to be limited by the following
explanation which is simply given to illustrate one current
plausible explanation of the results obtained with the present
invention. In accordance with the illustration of FIG. 22, the
sample rapidly propagates through the conduit 26 and into the
reaction volume 66. The liquid front advances swiftly, filling the
reaction volume, solubilizing the reagent 125 and initiating the
coagulation reaction. The light intensity (FIG. 22) as detected by
the photodetector 121 is initially high at the level indicated at
160. This is presumed to be because the dried reagent is highly
refractile. Upon addition of sample at a particular point in time,
162, the light intensity drops precipitously as shown at 164 due to
dissolution of the reagent, which apparently provides an improved
refractive index match. Elimination of air as the sample moves into
the reaction space probably contributes significantly to matching
of refractive indices. The decreasing scatter curve may then level
off before the beginning 166 of a discernable rise 168 is observed.
This rise may be due to the increase in scattered light intensity
arising from the polymerizing system which at or near this point in
time has formed a gel clot. The scatter intensity continues to
increase more slowly, 170, eventually levelling-off.
[0171] The prothrombin time is equal to or highly correlated with
the time elapsed between sample addition 162 and the clot formation
endpoint 166. Prothrombin time (interval 172) is shown below the
abscissa as 15 seconds (for the particular assay illustrated). It
is believed that this is the first successful example of a
prothrombin time determination being performed via light scatter
measurements with dry reagent.
[0172] FIG. 23 shows a simplified systems block diagram of how the
analog signal in FIG. 22 may be interpreted. The light source 120
transmits light through the reaction slide 1. Light scatter at
90.degree. is monitored by the photodetector 172 and amplified at
176. Digitizing is accomplished at 178, and peak and slope
detection are accomplished in block 180. At 180 start time and
endpoint detection are determined, as well as kinetic curve
characteristics. The resultant digital information is sent to the
microprocessor CPU, 182, which has other inputs and outputs 184.
Block 186 contains data and program memories. The results are read
on the display 188. In addition to monitoring the assay kinetics,
the dynamics of sample entry into the reaction space and initial
interaction with the reagent are monitored, as well, as a
consequence of the geometry and structure of the reaction slide.
The initial fall of the curve therefore provides information for
quality control of proper sample addition.
[0173] The above is an example of a measurement made by light
scattering. There will now be described an example of a measurement
made by reflectance. In this particular example, changes in
reflectance are used to monitor viscosity changes during a
coagulation reaction. Shown in FIG. 75 is a reaction slide 1
disposed above and in close proximity to a permanent magnet 195.
Beneath the permanent magnet 195 is an electromagnet 196 which is
driven by a power supply 199 for cycling voltage on and off at a
desired frequency. There is also provided a light source for
providing incident light and a detector positioned for detecting
light rays reflected from the sample within the reaction volume
66.
[0174] The reflected rays illustrated as rays 198 are detected by a
dector 400. Detector 400 can be positioned at any position between
the 90.degree. and the 10.degree. positions, inclusively, shown in
FIG. 75. Preferably detector 400 can be positioned between
90.degree. and 45.degree., most preferably between 90.degree. and
75.degree.. A measurement that has successfully been conducted with
such apparatus will now be described.
[0175] The reaction slide was prepared in advance by forming a
slurry of a coagulation reagent and inert magnetic particles
suspended in the reagent. The coagulation reagent was
thromboplastin-calcium, and the magnetic particles were magnetite.
Inert magnetic particles work well when provided in the range from
approximately 5 to 50 milligrams per milliliter of liquid reagent.
The slurry was applied to the reaction slide and freeze dried.
[0176] To perform the assay, the reaction slide 1 was introduced to
the apparatus in position as shown in FIG. 75. The light source was
a light emitting diode, and the detector was a silicon photodiode.
A chart recorder was AC-coupled to a photodiode amplifier. The
permanent magnet 195 was in the form of a sheet (which may be made
of a flexible or rigid magnetic material). The power to the
electromagnet was cycled at a frequency of 1 Hertz. A sample of
blood plasma was introduced into the sample well 64 and filled the
reaction volume 66, solubilizing the dry reagent, resuspending the
magnetic particles as shown at 197, and iniating the coagulation
reaction. The permanent magnet 195 causes the magnetic particles to
be drawn downwardly and lie down against the base 30 in an
orientation parallel to the plane of the permanent magnet. However,
each cycle of energy supplied to the electromagnet 196 causes the
magnetic particles to stand upright like tiny whiskers in an
orientation of alignment along vertical field lines. At the end of
each such energy cycle, the particles lie flat again.
[0177] The detected reflected light 198 shows a time-varying
pattern of light intensity in accordance with the above-recited
changes in orientation of the magnetic particles. The light
intensity is less when the particles lie flat than when they stand
upright.
[0178] The detected light intensity shows an initial peak at sample
addition. Thereafter, the detected light intensity cycles between
maximum and minimum values in accordance with the frequency of the
cycling of the electromagnet 196. During the period before clot
formation, the difference between the maximum and minimum values of
the detected light intensity increases. However, the peak-to-peak
light intensity oscillations begin to fall off from their maximum
values when a clot has started to form. At this point, the endpoint
has been reached. In the case of prothrombin, the elapsed time
between the sample addition peak and clot formation or clot onset
(endpoint) is easily measured. Resolution may be increased by
increasing the oscillation frequency.
[0179] For determining prothrombin time, the above-described
approach works extremely well using whole blood as well as plasma.
It is expected to work well for other types of blood coagulation
assays. The measurement may be made using transmitted light instead
of the described method of using reflected light. However, it is
thought less convenient to use transmitted light than reflected
light.
[0180] The above provides one example of a light scattering
measurement followed by one example of a light reflecting
measurement. Alternative means for introducing light into the
reaction volume 66 for such measurements will now be discussed
together with a discussion of other types of optical measurements.
In particular, there will be discussed optical measurements based
on transmission/absorbance, chemiluminescence, reflectance,
fluorescence, and combinations of these techniques.
[0181] Transmission/absorbance, or optical density measurements,
involve measurement of the ratio of light intensity before and
after light passes through a sample in the absence of (or
excluding) scattering. The Beer-Lambert Law describes the
phenomenon. Standard detectors are employed in a "line of sight"
configuration with the light source, as shown in FIG. 25.
Incandescent or LED light sources may be used.
[0182] FIG. 25 also illustrates an alternative means of introducing
light into the reaction slide and of measuring light that has left
the reaction slide. In particular, there are provided two external
waveguides 190, 191 which respectively carry incident light to one
of the internal waveguides 27 of the reaction slide 1 and receive
light that has been passed through the other internal waveguide 27
and channel the received light to the photodetector 121. The
external waveguides 190, 191 may be made of the same types of
materials used to produce the overlay 20 of a reaction slide, as
described above. Accordingly, it may be seen that use of an
external waveguide or waveguides 190, 191 provides structure for
introducing light into the reaction slide 1 that is alternative to
the polymeric housing 130 illustrated in FIG. 19.
[0183] An optical filter 192 may be used for wavelength
selection.
[0184] In FIG. 25, colorimetric or turbidometric measurement is
achieved. Light rays pass through filter 192, travel through
external waveguide 190 and through an internal waveguide 27, then
illuminate reaction volume 66. They pass through the reaction
volume, through the internal waveguide 27 at the right, and are
transmitted through optional second external waveguide 191. The
light rays are then directed to an appropriate detector 121.
[0185] In FIG. 26, a second light detector 121 for detecting
scattered light and an aperture 194 to restrict detection to light
scattered at or near 90.degree. have been added to the arrangement
of FIG. 25. Aperture 194 is analogous to aperture 154 of FIG. 22.
FIG. 26 therefore illustrates an embodiment which allows
simultaneous detection of scatter and absorption. It is based upon
a combination of light paths 40 and 41 in FIG. 4. This monitoring
strategy may be useful during the formation of precipitates or
large polymers with characteristic absorption spectra.
[0186] FIG. 27 shows an embodiment employing a reaction slide 1
with a transparent base and detector 121, but no light source other
than that initiated by a chemiluminescent reaction taking place
within the reaction space. The chemiluminescent reaction can be
triggered upon the addition of sample.
[0187] FIG. 28 shows an embodiment based upon reflectance. A
partial integrating sphere 200 mounts a light source 120 and a
light detector 121. The partial integrating sphere is positioned
beneath the base 30 of a reaction slide 1 having a reaction volume
66 and cover 10. Rays reflected back into the partial integrating
sphere from within the reaction slide are detected to allow
measurement of the reaction. It should be noted that the spacer 60
is not employed to provide internal waveguides for transmission.
Internal waveguides are not necessary for the type of measurement
performed in FIG. 27, either, but they can of course be used, if
desired, by positioning a detector so as to intercept light exiting
through an internal waveguide.
[0188] More generally, such reflectance measurements capture light
reflected in any desired direction from surfaces or surface layers.
A photodiode or photo-conductive cell may be used along with a
filter for wavelength specificity. The Kubelka-Monk Theory is a
useful model for reflecting systems.
[0189] A further method of detection may be based upon fluorescence
and involve the use of materials which are fluorescent and hence
absorb ultraviolet light and emit light of a longer wavelength,
frequently in the visible range. Fluorimetry may be used in a
reflective mode, similar to photodensitometry, for example to
quantitate samples on chromatograms. Variations are possible,
together with the use of fluorescence in combination with other
modes of detection. For example, a detector may be placed at a
fixed angle, typically 90.degree., from the direction of
transmission through a sample, as in nephelometry. As will now be
described in connection with FIGS. 29 and 30, fluorescence also may
be detected using an evanescent wave, for example at a solid/liquid
interface. Light paths such as those shown at 42 and 43 in FIG. 4
may be used.
[0190] FIG. 29 illustrates how a reaction slide 1 may be used to
perform fluorescent evanescent wave measurements near a wall
(cover/liquid interface) and alternating with or simultaneous with
colorimetric (or fluorescent) measurements through the reaction
volume 66. A first prism 202, which typically is not part of the
reaction slide but rests on top of the cover 10, is affixed to and
mechanically supported by first external waveguide 190 which, in
turn, rests atop the base 30 of the reaction slide 1. Fluorescent
light rays 42 are refracted by prism 202 and emerge, entering the
cover 10 at an angle somewhat greater than the critical angle. The
rays 42 undergo total internal reflection within the cover 10, as
shown at 206, and produce intense evanscent waves at the interface
between the cover 10 and the liquid in the reaction volume 66. The
evanescent waves excite fluorescent molecules bound at or near the
wall as a result of selective binding (or, depletion) associated
with an assay. The total internal reflected light then passes out
of the cover and is removed by second prism 204 situated atop
second external waveguide 191, passing out of the system.
Fluorescent emission of light is detected by a detector 121
positioned beneath the transparent base of the reaction slide.
[0191] Light ray 40 is sent into the system through external
waveguide 190 and internal waveguide 27, to illuminate the reaction
volume 66. It emerges through the opposite internal waveguide 27
and second external waveguide 191, exiting to be detected by a
detector 121 positioned laterally of the reaction slide.
[0192] Illumination rays 40 and 42 would typically be of the same
or similar wavelength but could excite molecules with different
emission spectra. In this figure, the detector 121 disposed beneath
the base 30 is shown to detect fluorescence emission resulting from
either excitation ray. Twin detectors could be utilized, as well.
Convection currents are created within the reaction volume 66,
using any technique to be described later.
[0193] The arrangement in FIG. 29 allows monitoring of the progress
of complex reactions involving bulk liquid species undergoing
reactions and simultaneous receptor site interactions at the
wall.
[0194] FIG. 30 shows another arrangement using evanescent wave
fluorescent detection. One application of this arrangement is rapid
measurement of hematocrit in a blood sample. In this case, prisms
202 and 204 rest beneath the base 30 of the reaction slide to
produce total internally reflected light 206 from the initial ray
43. Spacer 60 is not used in this example to transmit light. A
detector 121 is positioned beneath the base 30 opposite reaction
volume 66. A highly soluble fluorescent dye is reversibly absorbed
to the cover wall or base inside the reaction volume. The dye may
also be contained in a highly porous space-filling soluble
hydrophilic polymer with capillary or Interstitial structure. The
fluorescent dye is chosen such that it neither enters nor adheres
to the blood cells and is compatible with the blood plasma. In
general, most fluorescent dyes with net negative charge in solution
will be suitable, since they will not bind to or adsorb to the
surfaces of red blood cells and platelets (which have negative
surface charge). The exceptions are dyes that can enter the red
cells. These exceptions may generally be rendered suitable by
covalent bonding to an appropriate larger molecule (e.g. dextran or
polyglycols).
[0195] When the blood sample enters the reaction volume 66, the dye
is rapidly dissolved in the plasma, the rapidity of dissolution
being aided by the flow dynamics through the tortuous paths in the
space filling soluble polymer and/or by electromechanical mixing
effect, to be described below. The fluorescent signal near the wall
therefore quickly approaches a steady state, indicative of the
concentration of fluorophore in the plasma. From this
concentration, the known volume of the dye (and polymer, if
employed) and the volume of the reaction volume, the plasma volume
and hematocrit can be readily calculated.
[0196] Fluorescent dyes which may be possible candidates for use in
the hematocrit determination application may include: rhodamine B,
berberine sulfate, ethidium bromide, methylene blue, thionine, and
others, such as cyanine dyes (e.g.,
3,3'-diethyloxadicarbocyanine).
[0197] The method of hematocrit determination by dye dilution is
old (Eric Ponder, Hemolysis and Related Phenomena, Grune &
Stratton, New York, 1948, pp. 51-53). It has been applied in
various forms, such as in a continuous flow analyzer (Oberhardt, B.
J. and Olich, J., U.S. Pat. No. 4,097,237). Recently the method has
been applied using evanescent-wave fluorescence in a capillary tube
(Block, M. J. and Hirschfeld, T. B., European Patent Application,
Publication Number 0128723; Application Number 84303759.9; filed
Jun. 5, 1984; published Dec. 19, 1984).
[0198] The present invention is an improvement in that it utilizes
a self-contained convective effect to rapidly dissolve or aid in
dissolving the dye, thereby yielding more rapid test results than
would be otherwise possible. Additional improvement is afforded in
terms of increase in the accuracy of the determination in the
laterally flowing system created by forced convection, to be
described more fully below. In this flowing system, when lateral
flow is alternatively established, blood cells (particularly
erythrocytes) will on the average tend to rise to the center of the
stream due to hydrodynamic lift forces. This removal of cells from
the excitation region of the evanescent wave, however brief,
provides an interval for eliminating cell interference artifacts in
the fluorescence measurement.
[0199] Various exemplary methods for inducing forced convection
currents within the reaction volume 66 of a reaction slide 1 will
now be described with reference to FIGS. 31-34. Such forced
convection currents promote rapid and thorough mixing.
[0200] In FIG. 31 is shown a fine paramagnetic mesh or screen 208
disposed within the reaction space. The screen 208 may be supported
by sandwiching it between the cover 10 and base 30. In the
embodiment shown, the lateral edges of the screen are pressed
against an overlay 20 by an upper adhesive layer. The mesh 208 must
be capable of undergoing translational movement or bending while
being confined within the reaction volume 66.
[0201] The mesh may be a metallized polyester screen having a
coating, for example, of nickel. Other embodiments are possible.
For example, the screen may be made of nylon or polyester, coated
with a dispersion containing magnetic iron oxide. In the
alternative, the screen itself may be made of iron or steel and may
be coated with a protective plastic coat such as an elastomeric
coating. As an alternative to a mesh, a solid flexible support may
be used, with a distributed array of magnetic particles such as
magnetite coated with inert polymer bound to the solid support.
[0202] An oscillating magnetic field is supplied by electromagnet
210 connected to an appropriate time-varying electrical power
source 212. Under the influence of this magnetic field, the mesh
undergoes mechanical oscillations which translate to the
development of continuous oscillatory flow in the liquid contained
within the reaction volume for as long as the driving signal is
applied. The induced flow can be used to: (i) maintain a steady
state level of convection in a reaction volume containing sample
and reagents; (ii) assist in rapidly dissolving a reagent; or (iii)
increase convection near the wall (cover or base) to facilitate
transport of species to bound reagents.
[0203] FIG. 32 shows an alternative arrangement for mixing. In this
figure, permanent magnet 214 is affixed to cover 10 and driven into
an up/down oscillation by electromagnet 210 supplied by an
electrical driving signal from 212. The cover 10 moves along with
the magnet 214 as essentially one unit, causing periodic
alterations in the volume of the reaction volume 66. The inflow and
outflow of liquid produces mixing. The mixing resulting here is
well suited for moving liquid in the vicinity of the cover/liquid
interface. To achieve this type of mixing, the cover 10 may be
fabricated from a thin paramagnetic material, obviating the need
for a separate magnet 214. If a separate magnet is used, it may be
doughnut shaped, or disc shaped. It may also be made of flexible
ceramic magnetic material. A similar arrangement also is
illustrated in FIGS. 29 and 30.
[0204] FIG. 33 shows an arrangement which provides yet another
alternative for producing and sustaining a controlled convection
within the reaction volume 66. In this case, a solenoid 216 is
employed, having a rod 218 and a coil which is driven by an
appropriate intermittent unidirectional or time varying current
source 220 to push rod 218 against and deflect the cover 10. The
solenoid may be spring-loaded to retract the rod upward after
cessation of the current.
[0205] In the alternative and as shown in FIG. 34, there may be
provided a projecting element 222 passing through a hole 224 in an
orbiting disk 226. Tension spring 228 biases projecting element 222
downwardly as shown in the drawing. Drive 230 initially moves disk
226 downwardly for contact between the projecting element 222 and
the cover 10 of a reaction slide. At such time, projecting element
222 pushes against cover 10 at a localized pressure point with a
force, the magnitude of which is governed by spring 228.
Thereafter, drive 230 rotates disk 226 about the axis thereof,
causing the localized pressure point to trace a circle on the upper
surface of the cover 10. The cover 10 accordingly experiences a
localized deflection that moves in a circular pattern, following
the position of the projecting element 222. Such localized
deflection of the cover 10 causes mixing in the reaction volume 66.
It has been found that a deflection of 0.005 inches may be produced
in a polycarbonate cover 10 by a force of 3 ozs., applied with a
projecting element 222 having a 0.100 diameter circular
cross-section and a rounded bottom.
[0206] Yet another approach to mixing (not shown) is to utilize a
cover fabricated from a piezoelectric material or containing a
piezoelectric material affixed thereto. In this case, motion of the
cover would be produced piezoelectrically by application of the
appropriate voltage.
[0207] As noted above, a reaction slide according to the current
invention may be used to conduct assays in which the results are
measured using non-photometric techniques. That is, in addition to
transduction of light energy (e.g., transducers of the
photoconductive, photodiode or photomultiplier type), the reaction
slide may employ other mechanisms for energy conversion. Additional
types of transducers that are applicable to a reaction slide
according to the current invention include calorimetric
transducers, electrochemical transducers and viscosity transducers.
Examples of these will be described with reference to FIGS.
35-37.
[0208] Shown in FIG. 35 is a transverse vertical cross-section of a
representative embodiment of a reaction slide according to the
current invention. Calorimetric transducer 232 such as a
thermistor, thermocouple or thermopile is mounted by the base 30 of
the reaction slide such that at least a portion of the transducer
232 is directly exposed to the reaction volume 66. Electric leads
234, embedded in the base 30, extend from the transducer 232 to
electric contacts 236 mounted on the base 30 in a known manner. For
example, electric contacts 236 may be a conventional thick-film
polymer having contained therein a conductive powder. Appropriate
instrumentation is attached to the electric contacts 236.
[0209] The calorimetric transducer 232 may be used to measure
temperature change or heat input or output in an isothermal system.
Thus, the heat generated during a chemical reaction may be
monitored and used to quantify analyte concentration using
information related to heat of reaction.
[0210] As explained in-more detail below, a reaction slide
according to the current invention may be configured to allow
liquid to enter and exit the reaction volume 66 at predetermined
times. In such a case, using a calorimetric transducer 232,
temperature may be measured before and after residence of the
sample in the reaction volume. During residence, a known amount of
heat may be transferred to the liquid, as by a resistive heater 238
in the base 30 of the reaction slide. From the temperature change
(before and after heat input) the specific heat capacity of the
liquid (and analyte concentration for a pure dissolved analyte and
moderate concentration) may be measured using well-established
procedures.
[0211] The use of electrochemical transducers will now be described
with reference to FIG. 36, which is a top view of a reaction slide
according to the current invention with the cover removed. First
and second electrodes 240, 242 are provided in the reaction volume
66 and are spaced from each other. In the embodiment shown, the
electrodes 240 and 242 are conductive circular regions deposited on
the base 30 using known techniques. An electric lead 244 extends
from each electrode to electric contacts 246 formed on the base 30
of the reaction slide, taking care that the lead from the inner
electrode 242 does not make contact with the outer electrode
240.
[0212] Such electrochemical transducers may be of the
potentiometric type (e.g., pH measuring electrodes). In such a
case, a voltage may be generated which is proportional to the
analyte species concentration.
[0213] Also, amperometric methods may be employed, for example
using vacuum vapor deposited gold and silver. A polarographic
system may be used, which is a current measuring system having two
or three electrodes, such as a reference electrode, working
electrode and measurement electrode. An example is an amperometric
potentiostat. Such a system may be used with the application of
various voltammetric sweep patterns, depending upon the analyte
species to be detected.
[0214] Conductivity methods also may be applied. In such a case, an
electrode in the presence of an analyte exhibits a change in
conductance or resistance. Examples of this type include electrodes
fabricated from low dimensional materials such as polyacetylene,
polypyrrole, and the like.
[0215] Enzyme electrode systems and antibody electrode systems also
may be used.
[0216] FIG. 37 illustrates the use of a viscosity transducer. There
is shown a strain gage 248 mounted on the cover 10 of a reaction
slide 1. Using the strain gage 248, the rate of bending or movement
of the cover 10 or, in the alternative, the rate of recovery from a
downward push imparted by a solenoid-actuated push rod 250 may be
measured. Changes in the viscosity of fluid in the reaction volume
66, such as occur in coagulation reactions within the reaction
space, may therefore be measured using viscosity monitoring.
[0217] Viscosity monitoring is useful with a type of measurement to
be described below in which there is established a constant flow of
liquid into and from the reaction volume 66. An increase in the
viscosity of the liquid within the reaction volume 66 results in
increased drag and in retardation of motion of the cover.
[0218] As an alternative to the use of a strain gage 248 in
viscosity measurements, there may be used a piezoelectric element
mounted on the cover 10.
[0219] As has been noted above, a reaction slide according to the
current invention provides for the storage of a pre-measured amount
of reagent. One manner of providing for the presence of a reagent
has already been described in which a liquid reagent is placed in
the reaction volume and then dried, such that the dried reagent
coats the interior surfaces of the reaction volume. Other methods
will now be described with respect to FIGS. 38-41 and 61. Shown in
FIG. 38 is a longitudinal cross-section of a portion of a reaction
slide 1 according to the current invention. There is shown a
reagent-containing layer 252 disposed on the base 30 in the region
of the reaction volume 66. If desired, the reagent-containing layer
252 may extend further to the left than shown, occupying the
regions of the tapering walls 25, the conduit 26 or even extending
into the sample well 64. Although the illustrated reaction slide is
of the type that vents laterally (that is, there is no vent opening
in the cover 10), a reagent layer 252 may be used with any
embodiment.
[0220] FIG. 39 is a fragmentary vertical cross-section taken in the
region of the reaction volume of FIG. 38 and illustrating a first
specific embodiment of a reagent-containing layer 252 as shown in
FIG. 38. In particular, the reagent containing layer 252 is in the
form of a reagent-containing gel 254.
[0221] FIG. 40 is a similar view, showing a second embodiment of a
reagent-containing layer 252. In particular, the layer 252
comprises an upper layer 256 of a thin porous hydrophilic
(semipermeable) membrane. The membrane 256 is attached to the
second layer 258, which is in the form of a liquid absorbing matrix
(LAM).
[0222] The embodiment shown in FIG. 40 is especially useful for
plasma separation when whole blood is used as the sample. Iii
particular, plasma is drawn through the membrane 256 and into the
LAM 258, where it is stored. Further details of such an assay will
be described later. Variations of this embodiment are possible,
according to which the layer 252 may comprise the LAM 258 having
thereon a thin coating of polymer to provide a finer pore structure
at the upper surface of the LAM, such that the upper skin of the
LAM performs the same function as performed by the membrane 256.
That is, the fine pores of the thin polymer coating exclude cells
but admit plasma. Alternatively, the entire layer 252 may consist
of a single layer of a fine pore sponge.
[0223] FIG. 41 is a fragmentary vertical cross-section of an
additional embodiment of a reaction slide according to the current
invention, taken in the vicinity of the reaction volume 66. This
embodiment also is useful where whole blood is the sample and is
used for initiating a reaction at a desired time after plasma
separation is achieved. A cavity 260 is formed in the base 30 of
the reaction slide. Disposed in the bottom of the cavity 260 is a
reagent-containing layer 262, on top of which is an inert annular
spacer 264. Disposed above the spacer 264 is a LAM 258, there thus
being formed a gap between the LAM 258 and the reagent-containing
layer 262. Plasma-separating membrane 266 is disposed in the
reaction volume 66 in contact with the LAM 258.
[0224] With whole blood in the reaction volume 66, plasma will be
drawn through membrane 266 and stored in LAM 258. After such
separation is achieved, a reaction may be initiated at any desired
time by manually or automatically (for example, as with a solenoid
driver) pushing down on cover 10 to cause contact between the LAM
258 and the reagent-containing layer 262. Such contact will
initiate a reaction at the desired time.
[0225] FIG. 61 is a longitudinal cross-sectional elevation of a
reaction slide according to the current invention, showing yet an
additional method of incorporating a reagent into a reaction slide.
Reaction volume 66 is filled with a permeable polymeric matrix 270
having a reagent distributed throughout the matrix. It is a matter
of choice whether or not the matrix 270 extends to the right as
shown in the drawing into the region of the vent 76 or all the way
to the left in the drawing as far as the sample well 64. The use of
such a dry porous space-filling reagent in the reaction volume
provides capillary or interstitial fine structure that is capable
of controlling the filling beyond the control provided by the
capillary flow ordinarily arising from the spacing of the cover 10
and base 30.
[0226] It will be noted that performing an assay typically begins
with the introduction of a sample into a sample well 64 of a
reaction slide. FIG. 42 illustrates an additional embodiment of a
reaction slide according to the current invention, the embodiment
being useful as a "processing device" for initiating an assay at
any desired time by depositing a sample into a sample well of
another reaction slide at the desired time.
[0227] A passage 272 extends from the lower surface of base 30
upwardly to a recess 274 of greater lateral dimensions. Disposed in
the recess 274 are a plasma separating membrane 266 and a LAM 258.
With whole blood filling the reaction volume 66, plasma passes
through membrane 266 and is collected in LAM 258. Either before or
after such separation, the slide shown in FIG. 42 is positioned
above a second reaction slide in which the assay is to be
performed. In particular, it is positioned such that the passage
272 lies directly above the sample well 64 of the second reaction
slide. At the desired time, the cover 10 of the slide shown in FIG.
42 is depressed, and such depression will result in the expulsion
of a drop or droplets of plasma from LAM 258, through passage 272
and into the sample well of the second reaction slide to initiate
the assay.
[0228] As has been discussed above, the various embodiments of
reaction slides according to the current invention are useful in
performing a variety of assays. A prothrombin time determination
has been described above. More specific examples are given below in
Examples 1-7. There will now be described what may be called a
continuous flow assay involving the separation of cells in a
flowing stream. The embodiments of FIGS. 38-41 are especially
useful for such assays. An important conceptual difference between
such assays and those in which the sample remains in the reaction
volume 66 (such as the prothrombin time determination) is that, in
a continuous flow assay, there is a comparatively larger volume of
sample available in the sample well 64, and a continuous flow is
established from the sample well 64 to the LAM 82. To this end, it
may be desirable to enlarge the available volume of the sample
well. FIG. 38 shows one way in which the sample well 64 may be
enlarged. In particular, annular sample well extender 276 is fixed
atop the cover 10 of the reaction slide 1 to increase the volume of
the sample well 64. Other means of accomplishing this result may
present themself to those of ordinary skill in the art.
[0229] A liquid sample is placed in the sample well 64. This sample
rapidly flows to the right in FIG. 38, filling the conduit 26 and
the reaction volume 66. Upon contact of the liquid with the LAM 82,
a flow condition is set up whereby the liquid is pulled in and
flows continuously into the LAM 82 until the sample well 64 is
exhausted.
[0230] The established laminer flow condition is advantageous for
analysis or processing of certain types of samples, such as samples
with suspended particles (including but not limited to whole
blood). A characteristic of the continuous flow is that suspended
matter moves away from the top, bottom and side walls of the
passage in which it flows and toward the center of the stream. Such
material accordingly does not participate to a substantial degree
in chemical, enzymatic or immunological reactions caused by
reagent-containing layers 252. Thus, the laminar flow condition
driven by the LAM 82 provides a separation step. Color development
may be read in the reagent layer 252 in a variety of ways, such as
the use of reflectance measurement technology illustrated in FIG.
28.
[0231] In a continuous flow measurement, when the sample is blood,
an embodiment similar to those described above in regard to FIGS.
40 and 41 is desirable. The pore structure used for passing plasma
and excluding blood cells should be such that the pores have a size
of approximately 1.2 microns or less. For the use of whole,
non-anticoagulated blood, anticoagulant may be present, if
required, in LAM 82 and possibly in the vicinity of or contained
within the reagent-containing layer 252.
[0232] Separation of cells (or particulates) in a flowing stream
from the medium in which the cells are suspended may be achieved
under the appropriate flow conditions, since the cells migrate away
from the walls of the conduit and toward the center of the stream.
As disclosed by B. A. Solomon (Membrane Separations: Technological
Principles and Issues, Vol. XXVII, Trans. Am. Soc. Artif. Organs
1981 pp. 345-350), blood flow sustained parallel to the plane of a
suitable membrane allows plasma to be drawn through the pores of
the membrane without damaging the cells, due to the tendency of
cells to migrate away from the membrane surface. In the present
invention, the sustained transverse flow is achieved not by pumping
or vacuum (pressure differentiation) induced by mechanical means
but through the use of a fine capillary or absorbant structure
(LAM) to remove liquid (e.g., blood) and thereby to cause movement
or flow in a direction parallel to the plane of the membrane. At
the same time, a capillary action through the membrane and into a
second Liquid absorbing matrix (LAM) is achieved to collect the
separated blood plasma on the opposite side of the membrane. The
reaction volume dimensions provide a conduit or chamber such that
the flowing liquid develops shear forces sufficient to minimize
passage of cells through the membrane and hence to minimize cell
destruction. The second LAM may contain reagents for performing an
assay. The second LAM may be utilized only for collection (and
could contain anticoagulants, preservatives, etc.). Diagnostic
reagents could be bound to the membrane or contained in another
layer adjacent to the membrane or LAM.
[0233] The art of removing a plasma sample from whole blood using a
separating matrix and later contacting it at a precise time with a
reagent layer has been described for use with a gel or porous
medium (Oberhardt, B. J., U.S. Pat. No. 4,288,228, Sep. 8, 1981)
and later (by Vogel, P., et al., U.S. Pat. No. 4,477,575, Oct. 16,
1984) for use with a glass fiber layer. The present invention shows
yet another way to achieve the objective of plasma separation and
reaction initiation at a precise time.
[0234] FIGS. 43-60 show how a reaction slide may be utilized to
perform an assay involving a bound reagent, several added reagents,
and washing steps. An ELISA type immunoassay and-the embodiment of
FIG. 6 are used as an example. In FIG. 43, a sample to be analyzed
is placed in the sample well. At the bottom of the reaction space
is a layer 252 of covalently attached antibody molecules 278. This
layer is better visualized in FIG. 52, to the right of FIG. 43. A
moveable sponge or LAM 51 which forms part of the disposable
reaction slide is situated on the base and fastened by any of a
variety of mechanisms to allow it to be moved by an external
mechanical driver to alternately engage and disengage the LAM from
the vent 76 of the reaction volume 66.
[0235] FIG. 44 shows the sample filling the reaction volume. FIG.
53 shows greater detail indicating the capture of antigen molecules
280 by the bound antibody 278. Some unbound antigen 282 and other
molecular species 284 are shown, as well. In FIG. 45, the LAM is
depressed to remove all liquid from the system and is subsequently
returned to its original position. Emptying the reaction volume of
liquid can be monitored electro-optically, as previously described.
In FIG. 54, the antibody molecules with captured antigen are shown.
Although not indicated, it is also possible to introduce into the
sample well a buffer liquid and then repeat the step shown in FIG.
45 to achieve a better washing. This washing step with buffer,
which is well known in the art, could be repeated, if
necessary.
[0236] In FIG. 46, reagent is introduced. This second reagent (the
first being the bound antibody) is rapidly drawn in (FIG. 47) and
consists of an antibody-enzyme conjugate 286. After a short
incubation (FIGS. 48 and 57), some of the antibody enzyme conjugate
molecules become bound 288 to a second site on the antigen molecule
(with different specificity than the first site) and some conjugate
molecules remain free 290. The liquid is then removed in FIG. 49
(as in FIG. 45) with the possible incorporation of additional
washing steps with a buffer. In FIGS. 50 and 59, a developer
reagent consisting of a fluorogenic or chromogenic substrate 292 or
combination of substrate and developer dye is added. FIG. 51 shows
the filling of the reaction volume, and FIG. 60 shows the
development of chromophores 294 or other optically detectable
species.
[0237] Thus, it is shown that the LAM-reaction slide can allow a
precise multistep immunoassay to be conducted with imprecise
pipetting steps and simplified wash steps. This could be combined
with forced convection to facilitate reactions in FIGS. 53, 56 and
59 (as shown in previous embodiments utilizing internal mixing). Of
major significance is that labor can be significantly reduced with
the use of simple and inexpensive instrumentation performing
operations on and interacting with the reaction slide.
[0238] As was the case with the embodiment illustrated in FIG. 13,
the configuration of the LAM as illustrated in FIGS. 43-51 may, if
desired, be modified by having the LAM overlap over part of cover
10. In this other embodiment, the LAM is situated above the base
and is in contact with the cover. Removal of fluid from the
reaction chamber is achieved by compressing the LAM down toward
base 30 so that. contact is established between the LAM and the
fluid in the reaction volume.
[0239] Shown in FIG. 62 is a top view of a first embodiment of a
reaction slide of a type having plural reaction volumes. Cover 10,
shown partially cut-away in the figure, is provided with openings
14, used in forming a plurality of sample wells 64, and rectangular
openings 12 forming vents 76 leading to the reaction volumes 66.
The spacer 60 may be any of the types previously described. In the
illustrated embodiment, there are provided in the spacer 60 a
plurality of sample receiving openings 22, which also form part of
the sample wells 64. Each sample receiving opening 22 communicates
with a reaction space 24, also cut from the spacer 60. As in the
preceeding embodiments, the reaction space 24 is used in
conjunction with cover 10 and base 30 to form a plurality of
reaction volumes 66. Tapered walls 25 are provided communicating
the sample receiving opening 22 and reaction space 24.
[0240] Disposed beneath the spacer 60 is a base 30, which is
visible through the sample receiving openings 22 and reaction
spaces 24.
[0241] A plurality of slots 302 are cut through both the spacer 60
and base 30 and disposed adjacent each reaction volume 66. The
slots 302 are configured to receive external optical waveguides
that extend upwardly through the base 30 and spacer 60 for carrying
light to and from the internal waveguides 27 formed between the
slots 302 and the reaction volumes 66. As above, such light may be
used for measurements using colorimetry, light scatter, and similar
measurements.
[0242] It may be seen that the embodiment 300 provides dense
packing to achieve more reaction spaces per unit area of common
base 30.
[0243] The embodiment 300 may be used with a single set of external
waveguides, one mixing station and one LAM actuating mechanism (not
shown), each of which may be indexed from space to space, as
required, to service designated reaction volumes 66. In the
alternative, there may be used a plurality of fixed external
waveguides, mixing stations and LAM actuators, and these may be
used independently or simultaneously.
[0244] As a variation, individual LAM's may be provided on a strip
extending from left to right in the drawing, such that depressing
the strip causes the LAM's to withdraw liquid from the vents 12 of
each reaction volume 66 in a single row. In FIG. 62, three such LAM
strips would be used, as there are three rows of reaction
volumes.
[0245] Samples may be added to the sample wells 64 independently or
simultaneously.
[0246] Shown in FIG. 63 is a top view of a second embodiment 304 of
a reaction slide having plural reaction spaces. There is provided a
solid base 30, a cover 10 having a sample receiving opening 14
formed therein, and a spacer disposed between cover 10 and base 30,
the spacer being formed of a U-shaped member 306 and two dividers
308. The elements 306 and 308 are of uniform height such that cover
10 is uniformly spaced from base 30.
[0247] The sample well 64 extends downwardly into the U-shaped
member 306 and communicates through passage 316 with each of three
reaction volumes 310, 312 and 314. Each of the three reaction
volumes vent laterally, which is toward the top of the drawing. If
desired, an external LAM 82 may be provided as shown in FIG. 13. A
separate LAM may be provided for each reaction volume, or a single
LAM may serve all three reaction volumes.
[0248] Addition of liquid sample to sample well 64 causes rapid
filling of all three reaction volumes. The center volume 312 fills
slightly faster due to its location adjacent passage 316. If
desired, the sample well 64 may be relocated, or other geometric
changes may be made to cause each of the reaction volumes 310, 312
and 314 to fill at the same rate.
[0249] Shown in FIG. 64 is a top view of a third embodiment 318 of
a reaction slide having plural reaction volumes. Cover 10 is
provided with a sample receiving opening 14, which forms part of
the sample well 64. The spacer 60, of uniform height and disposed
between cover 10 and base 30, is provided with a cut-out forming
three reaction volumes 320, 322 and 324. Reaction volume 320
communicates with sample well 64 through passage 326, also cut into
the spacer 60. Passage 328, cut into the spacer 60, communicates
reaction volume 322 and reaction volume 320. Passage 330
communicates reaction volume 324 and reaction volume 322. The
distal end 332 of the spacer 60 is open, such that reaction volume
324 vents laterally, to the right in the drawing. A LAM may be
provided as shown in FIG. 13.
[0250] It may be seen that the reaction volumes fill sequentially
from sample well 64. When it is desired to remove sample from the
reaction volumes, a LAM applied to the distal end 332 of the spacer
60 will first empty reaction volume 324. It has been found that, if
the LAM is suddenly removed, it is possible to bring about the
stepwise transfer of sample from reaction volume 320 into reaction
volume 322 and the simultaneous movement of sample from reaction
volume 322 into reaction volume 324. Such action may be used to
facilitate sequential rections through the translation of contents
of reaction volumes laterally through any desired number of
sequential reaction volumes.
[0251] FIGS. 65 and 66 show a fourth embodiment 336 of a reaction
slide according to the current invention, the reaction slide 336
having plural reaction volumes filled by parallel filling. Among
the uses of reaction slide 336 is that it is useful in conducting a
Plasminogen Activator assay. Also shown in FIGS. 65 and 66 is
instrumentation useful in conducting the TPA assay.
[0252] In reaction slide 336, the base 30, the spacer and the cover
10 have been cut out as shown at 338 to form first and second legs
340, 342. The cover 10 is provided with an opening for the sample
well 64 and openings for each of the vents 354 that communicate
respectively with first reaction volume 350 and second reaction
volume 352. The spacer is cut out to form the sample well 64 and to
form common conduit 344 which branches to form first and second
branched conduits 346, 348, the branched conduits respectively
leading to the reaction volumes 350 and 352. As in certain of the
previous embodiments, the spacer is transparent to provide internal
waveguides 27 adjacent the reaction volumes. It will be seen that a
sample placed in sample well 64 will be drawn by capillary action
through common conduit 344 and will then divide, proceeding through
branched conduits 346, 348 and into the reaction volumes 350,
352.
[0253] Also shown in FIGS. 65 and 66 are first light source 356,
second light source 358, first scatter detector 360, second scatter
detector 362 and transmission detector 364. Light shield 366
protects the second reaction volume 352 from receiving radiation
from first light source 356. It may be seen that light from first
and second sources 356, 358 respectively enters first and second
reaction volumes 350, 352, where some of it is scattered at
90.degree. and passes through base 30, whereafter the scattered
light is detected at 360 and 362. Transmission detector 364 detects
that portion of the light from source 358 which has been neither
scattered nor absorbed in reaction volume 352.
[0254] The various embodiments of reaction slides according to the
current invention may be provided with apparatus for accomplishing
"selective flow", according to which the filling of a reaction
volume from a sample well may be delayed for any desired length of
time after a sample has been placed in the sample well. Such
selective flow may be accomplished by selective venting, as will be
described with reference to FIG. 67, or by use of a pinch valve, as
will be described with reference to FIGS. 68 and 69.
[0255] In selective venting, capillary action is initially
prevented by blocking the downstream egress of air. This is most
easily accomplished in those embodiments of a reaction slide in
which the venting is accomplished vertically, through an opening
through the cover. FIG. 67 is a vertical longitudinal cross-section
of a representative reaction slide 1. The reaction slide 1 includes
a vent cover assembly 370, the vent cover 370 including a cover
support 372 and an elastomeric pad 374 attached to the pad 374 is
not a LAM and the cover support 372. Elastomeric pad 374 may be
made, for example, of silicone or latex. Structurally, the cover
support 372 is similar to the LAM support 52 described above in
relation to FIG. 6, except that the pad 374 is not a LAM and the
cover support 372 continually biases the pad 374 against the vent
opening 12, blocking it.
[0256] A sample placed in sample well 64 will remain in the sample
well until such time as the pad 374 no longer blocks the vent. Such
unblocking of the vent may be brought about by lifting cover
support 372. Such lifting may be accomplished manually,
mechanically or electromechanically.
[0257] Modifications are possible. For example, the cover support
372 need not be attached to base 30 but may, instead, take the form
of a straight element extending to the right in the figure. Any
appropriate means may be used to grasp the support 372 and lift it.
In such a case, it may be desirable to provide a slight adhesive
bond between the pad 374 and cover 10 to prevent premature
displacement. In the alternative, a vent cover assembly may be
contained in an instrumentation housing, completely separate from
the reaction slide 1. When the reaction slide is inserted into the
housing, the vent cover assembly may be brought downwardly to cover
the vent.
[0258] FIGS. 68 and 69 illustrate a pinch valve mechanism.
Elastomeric disk 376 is fixed in an opening in cover 10, the disk
376 being disposed directly above conduit 26 which connects the
sample well 64 and the reaction volume 66. Pinch rod 378, which may
be attached to a control mechanism in an instrumentation housing,
is vertically movable so as to descend and press the elastomeric
disk into the channel 26, thereby blocking flow from the sample
well 64. At any desired time, the pinch rod 378 may be raised to
allow the sample to reach the reaction volume 66.
[0259] With the incorporation of selective flow by selective
venting or a pinch valve mechanism, or both, it is possible to send
a liquid or liquids to a reaction volume at a predesignated time or
to move liquid streams out of one reaction volume and into another
at a predesignated time, as described below. It also is possible to
stop the flow of liquid into a reaction volume, for example, when
it is partially filled. Among the possible applications are as
follows:
[0260] A. A chemical reaction may be initiated at a specific time,
such as after a prior reaction has taken place (e.g., for
diagnostic assays).
[0261] B. Mixing of two liquids is possible and easy to control, as
also described below.
[0262] C. As described below, cascading of reaction spaces may be
controlled.
[0263] D. By mixing two liquids, it is possible to perform
dilutions (sequential dilutions).
[0264] FIG. 70 is a fragmentary top view of a reaction slide in
which an interconnecting conduit 380 is formed in the spacer. In
the drawing, the left end of conduit 380 communicates with reaction
volume 66. The conduit extends to the right for transferring liquid
from reaction volume 66 to one or more additional reaction volumes
disposed on the same base 30. FIG. 71 schematically illustrates one
use for such an interconnecting conduit 380.
[0265] As schematically represented in FIG. 71, a reaction slide
may comprise first and second sample wells 382, 384, first and
second reaction volumes 386, 388 respectively fed from the sample
wells 382, 384, and third reaction volume 390 fed by two
interconnecting conduits 380 which respectively communicate with
the reaction volumes 386, 388. All of these elements may be a part
of the same reaction slide. That is, all of them may be disposed on
a common base 30 and formed by a single appropriately-cut spacer.
Elements 392, 394 and 396, functionally and schematically
represented as valves, may physically take the form of three
separate vent cover assemblies for selective venting as shown in
FIG. 67.
[0266] According to FIG. 71, two separate liquids may first be
introduced into reaction volumes 386 and 388 from their respective
sample wells 382, 384, at any desired time. Thereafter, the two
liquids are mixed in volume 390 for further reaction or measurement
or both.
[0267] For example, with vents 392, 394 and 396 closed, sample well
384 may be filled with a sample and sample well 382 may be filled
with a reagent. Dry reagents are disposed in the reaction volumes
386, 388 and 390, having been put there previously. At a desired
time, vents 392 and 394 are opened to allow the reaction volume 386
to fill with reagent from sample well 382 and to allow the reaction
volume 388 to fill with reagent from 384. Thereafter, incubation or
mixing or both occurs in reaction volumes 386 and 388, for a
preselected time through the use of previously-described means.
Then, with vents 392 and 394 closed, vent 396 is opened to draw
fluid through the two interconnecting channels 380 into reaction
volume 390. The reaction volume 390 may have a volume equal to or
less than the combined volumes of 386 and 388. Mixing may be
carried out in 390, and a reaction therein may be monitored by any
of the previously-described means for monitoring a reaction. If
desired, reaction volumes 386 and 388 may similarly be
monitored.
[0268] If desired, the two interconnecting conduits 380 may be
dimensioned such that they compensate for differences in viscosity
between the liquids and the two reaction volumes. For example, if
the liquid in volume 386 is more viscous than that in volume 388,
and if it is desired to mix both liquids in equal proportions in
390, then the upper interconnecting conduit 380 in the drawing may
be made wider than the lower interconnecting conduit 380 to produce
substantially equivalent flow under capillary flow driving forces
in the system. Light scatter sensors may be used to monitor the
three reaction volumes to determine when the volumes are filled or
empty, to determine how fast a volume is filling, and to operate
controls to open and close the vents to start and stop the
flow.
[0269] It will be understood that the same results may be
accomplished using another means for achieving selective flow. For
example, pinch valves may be incorporated both upstream and
downstream of each of the reaction volumes 386, 388.
[0270] FIG. 71 is a simple example of cascading, more complex
examples of which will now be described.
[0271] FIG. 72 shows four levels of cascading, which will be
referred to as level I, level II, level III and level IV. Level I
is represented by reaction volumes 408, 410, 412 and 414. Each of
these reaction volumes is respectively supplied from an associated
sample well 400, 402, 404 and 406. Reaction volumes 408 and 410
feed reaction volume 416 through interconnecting conduits 380.
Similarly, reaction volumes 412 and 414 feed reaction volume 418
through an additional set of interconnecting conduits. Selective
flow is provided by pinch valve mechanisms 426. Reaction volumes
416 and 418 represent level II. By similar structure, reaction
volumes 420 and 422 represent level III, and reaction volume 424
represents level IV. If desired, additional reaction volumes may be
provided as indicated by the dashed line extending to the left of
reaction volume 422 in the drawing. The size of the reaction volume
at level IV is typically, but not necessarily, twice as great as
the combined volumes of the reaction volumes at level III.
Similarly, the combined volumes of the reaction volumes at level
III are typically two times as great as those at level II. Each
reaction volume may be empty or may contain a dry reagent. Any or
all reaction volumes may be subjected to mixing by
previously-described means and may be configured for liquid removal
by a LAM. Any or all reaction volumes may be monitored by
previously-described methods and also inspected by automated light
microscope or by eye.
[0272] At level I, each of the liquids may be reacted and studied
or monitored. A decision may be made thereafter to move any or all
of the liquids to level II. Depending upon the nature of the
decision made, further measurement may be made in reaction volume
416, 418, or both, where a further reaction may be monitored.
[0273] More specifically, if the contents of the sample wells
400-406 are respectively representated as .alpha., .beta., .gamma.
and .DELTA. any or all of these liquids may be reacted at level I.
Thereafter, at level II, .alpha. and .beta. may be reacted together
in reaction volume 416. In the alternative, .alpha. may be sent
alone to level II and reacted with dry bound reagent in reaction
volume 416. The liquid may then be absorbed by a LAM to empty the
reaction volume 416, after which time may be admitted to reaction
volume 416 to further react with products captured by the bound
reagent in reaction volume 416. Similar procedures and decisions
may be made with regard to samples .gamma. and .DELTA..
[0274] The resultant product in reaction volume 416 may be sent to
reaction volume 420, either alone or in admixture with a liquid
from reaction volume 418. The process then continues analogously
until reaction volume 424 is filled with liquid from reaction
volume 420 or from 422 or from both.
[0275] FIG. 73 schematically illustrates cascading in an order that
is inverse from that shown in FIG. 72. In particular, a single
initial sample placed in sample well 428 and initially reacted in
reaction volume 430 may later be channeled to either or both of
downstream reaction volumes 432, 434, and so forth. Selective flow
in FIG. 73 may be accomplished by pinch valve mechanisms or by
selective venting, as described above.
[0276] Yet a further example of cascading is shown schematically in
FIG. 74, in which a plurality of sample wells and associated
reaction volumes selectively feed a single reaction volume 436, and
the product of a reaction in the volume 436 may, if desired, be
further sent to yet additional reaction volumes, is illustrated by
the interconnecting channel 380 shown in phantom. Such additional
reaction volumes may be disposed in serial, in parallel or
cascaded.
[0277] In general, in regard to FIGS. 70-73, it should be noted
that the sizes of the sample wells and reaction volumes should be
such as to provide a sufficient volume of liquid to fill whatever
volumes are anticipated will be needed. For example, in FIG. 73, it
may be necessary for the volume of sample well 428 to be sufficient
to fill each of the illustrated reaction volumes. One means of
providing such a sufficient volume of sample has been described
above in the form of the enlarged sample well of FIG. 38.
[0278] Thus, it may be seen that cascading of reaction volumes
allows reactions to be initiated and monitored and, based upon the
results obtained and decisions made (e.g., made by computer),
cascading further allows for additional or subsequent reactions to
be initiated, controlled and monitored using reaction products from
the first reactions or samples from the first reactions. This
provides the capability of performing complex assays with simple
apparatus. It also allows a "tree assay" to be performed. That is,
it allows for a series of branched assays to be performed in
parallel fashion but connected via branch structure to a "tree" to
answer a specific question. For example, the first reaction volume
could answer the question: "acid or base?" The sample or reaction
products could then be sent to one of two reaction volumes, one to
perform further tests on "acids" and one to perform further tests
on "bases", and so forth. Ultimately, highly specific information
may be known about the analyte.
[0279] Another application might be in finding an antibody or
antigen molecule which is specific for a given antigen or antibody.
In this case, antibodies or antigens may be placed (or bound) in
the reaction volumes, and the appearance of reactivity may be
monitored and used as a basis for selecting other reactions.
Similar applications could apply to DNA, DNA-probe reactions,
enzyme-substrate reactions and so forth.
[0280] Specific examples will now be given disclosing the use of
various embodiments of a reaction slide according to the current
invention. Other features of this invention will become apparent in
the course of these exemplary embodiments which are given for
illustration of the invention and are not intended to be limiting
thereof.
Example 1
[0281] A 0.020 inch thick (3.0.times.0.75 inch) strip of
polycarbonate was prewashed with distilled water and dried. Upon
this strip or base was placed a two-layer spacer consisting of two
pieces of a double-sided tape made from two thin sheets of
unplasticized polyvinyl chloride, each sheet previously having been
coated on both sides with medium-firm pressure sensitive acrylic
adhesive. The two-layer overlay had previously been cut out in the
center to create a sample well, conduit, reaction space, and vented
area, as may be seen in FIG. 5. The double overlay composite plus
adhesive coatings was of sufficient thickness to provide a final
spacing between the base and cover of 0.007 inches. The cover was
cut from 0.010 inch precleaned polycarbonate sheet and included a
rectangular vent hole and a circular sample well hole. The cover
was placed on top of the overlay and pressure applied by means of a
small roller to join the overlay to the cover and base.
[0282] The reaction volume was filled by means of a small syringe
fitted with a 25 gauge needle and positioned so that the solution
could flow from the edge of the sample well into the reaction
volume. The solution in the syringe was a conventional rabbit brain
thromboplastin emperically diluted to an appropriate concentration
for use in manual or automated prothrombin time determinations.
(Reagent calibration was previously checked by means of a standard
manual prothrombin time test using control plasma.) The reaction
slide was then frozen at -70.degree. C. and freeze dried.
[0283] A blood sample was collected by venipuncture using a 21
gauge needle. An evacuated tube containing sodium citrate, final
concentration 3.8 mg/ml was used for collection. The first of two
tubes was discarded, in accordance with NCCLS guidelines. The
second tube was centrifuged in a conventional blood bank centrifuge
(Clay Adams Sero-Fuge) and the plasma decanted. One drop of the
plasma sample was placed in the sample well of the reaction slide
(after the reaction slide was placed in light scattering
measurement apparatus as shown in FIGS. 20 and 21 and prewarmed to
37.degree. C. by means of a feedback controlled surface heater).
The plasma sample immediately flowed into the reaction volume. The
starting point, as monitored by the scatter detector, was clearly
defined, and the endpoint was clearly discernable. This allowed the
elapsed time to be readily measured and used to determine
prothrombin time (as a percent of control plasma value).
[0284] In this case, the light source was a high power light
emitting diode (635 nm gallium arsenide/gallium phosphide) and the
detector was a cadmium sulfide photoconductive cell. The photocell
output was connected to a resistive bridge circuit and a chart
recorder. Chart speed was 1 mm/sec, and gain was set at 1 volt full
scale. Bridge voltage was 18 volts.
EXAMPLE 2
[0285] A reaction slide was prepared as indicated in Example 1
except that it was fabricated using a single sheet of double sided
tape as an overlay material (total thickness 0.0035 inches). The
reaction slide was tested identically in the same instrument and
provided a similar endpoint but with reduced signal intensity and
longer lag phase (descending portion of the curve).
EXAMPLE 3
[0286] A reaction slide prepared as indicated in Example 1 was
placed in the monitoring instrument described in the previous
example. A sample of citrated whole blood was added to the sample
well. The resulting light scatter curve was observed to show an
initial transient increase in scatter intensity, establishment of a
new steady state level, and an eventual gradual rise from that
level at the onset of a clot. Although the curve was different, the
endpoint was similar but with reduced signal intensity. Elapsed
time was equivalent to that observed for the plasma curve.
EXAMPLE 4
[0287] A reaction slide prepared as indicated in Example 1 was
placed in the monitoring instrument described in that example.
Samples of citrated whole blood obtained from nine patients
undergoing anticoagulant therapy (from a collaborataive study with
Memorial Hospital, University of North Carolina, Chapel Hill) were
centrifuged, and the plasma samples decanted and tested. A
comparison was made of the values obtained with the light scatter
instrument and reaction slide versus the clinical laboratory values
obtained with a General Diagnostics Coag-A-Mate X-2 Automated
Coagulation Analyzer. Although the actual elapsed time values are
different for both instruments, an excellent correlation (0.95) was
obtained. The endpoints were extremely sharp for the reaction slide
values, and the clinical laboratory values could be calculated
easily from the reaction slide values by using a conversion
factor.
EXAMPLE 5
[0288] A reaction slide prepared as indicated in Example 1 was
placed in the monitoring instrument also described in Example 1. A
finger stick was performed using an Autolet.RTM. Automatic Lancet
(Ulster Scientific, Inc., Highland, N.Y.). One drop of blood was
placed on the slide sample well. The blood was drawn immediately
into the reaction space, initiating the reaction. The prothrombin
time endpoint was discernable from the scatter curve in the form of
a slope change. As in the case of the previous whole blood example
(Example 3), the absolute magnitude of scatter was different
(greater) for whole blood than for plasma. However, the elapsed
time, and consequently the test result, was essentially the
same.
EXAMPLE 6
[0289] a) Reaction slides were fabricated employing spacers as
described in Example 1, but with 3, 4, and 5 overlay layers to
increase spacer thickness and conseqeuently chamber height of the
reaction volume to 0.0105, 0.0140, and 0.0175 inches, respectively.
It was observed that reaction slides with reaction volume heights
of greater than 0.015 inches were not as reliable for retaining
samples and aqueous reagents via capillary forces without
leakage.
[0290] b) It was similarly observed that reaction slides prepared
as in Example 1 with spacers less than 0.0015 inches thick tended
to be difficult to fill and empty of liquid.
[0291] c) A reaction slide prepared as in Example 1 but with
cyanoacrylate adhesive and a 0.002 inch thick polyethylene
terephthalate sheet did not work well. (Two cyanoacrylate
preparations were tried: Wonder-Bond Plus, Borden, Inc., Columbus
Ohio and Pacer Tech Advanced Technology Series ATS-HC5, Pacer
Technology & Resources, Campbell, Calif.). Waveguide properties
were hampered by inhomogeneities in the adhesive, and
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