U.S. patent number 3,873,268 [Application Number 05/279,069] was granted by the patent office on 1975-03-25 for multiple solution testing device.
This patent grant is currently assigned to Pfizer Inc.. Invention is credited to James E. McKie, Jr..
United States Patent |
3,873,268 |
McKie, Jr. |
March 25, 1975 |
MULTIPLE SOLUTION TESTING DEVICE
Abstract
Drops of different solutions suspended from the perforations of
an upper plate are moved down into contact with a suitable reactive
layer or substrate disposed on a lower plate, or the drops may
bulge above a welled lower plate which is contacted with an upper
reactive layer. The reactions between each of the solutions and
reactive layer simultaneously commence when the suspended or
upwardly bulging drops contact the reactive layer. A sheet of
unexposed but developed photographic film incorporating at least
one gelatin layer provides a common reactive layer for comparing a
number of trypsin-catalyzed reactions. The film is conveniently
handled and inspected when attached to an indexed glass plate. The
photographic film is immersed in a low pH bath to simultaneously
terminate and preserve a record of all of the inherently concurrent
reactions. A simple press with a removable cavitied plate and
reactive layer advantageously performs the contact.
Inventors: |
McKie, Jr.; James E. (Ledyard,
CT) |
Assignee: |
Pfizer Inc. (New York,
NY)
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Family
ID: |
26856304 |
Appl.
No.: |
05/279,069 |
Filed: |
August 9, 1972 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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159795 |
Jul 6, 1971 |
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Current U.S.
Class: |
436/169; 422/504;
422/948 |
Current CPC
Class: |
B01L
3/5025 (20130101); G01N 31/00 (20130101); B01L
3/0289 (20130101); B01L 2200/0642 (20130101); B01L
2400/022 (20130101) |
Current International
Class: |
B01L
11/00 (20060101); B01L 3/00 (20060101); G01N
31/00 (20060101); G01n 031/20 () |
Field of
Search: |
;23/23B,23R,253R
;424/11,12 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Reese; Robert M.
Attorney, Agent or Firm: Connolly and Hutz
Parent Case Text
CROSS-REFERENCES TO RELATED APPLICATIONS
This application is a continuation-in-part of copending application
Ser. No. 159,795, filed July 6, 1971, now abandoned.
Claims
I claim:
1. A method of simultaneously testing the reactions of a number of
solutions with a reactive layer which comprises the steps of
placing substantially equal quantities of each of said solutions in
cavities in a holder whereby substantially equal quantities of said
solutions are caused to project substantially equal distances from
said holder, disposing said reactive layer upon a plate, aligning
said holder and said plate parallel to each other whereby each of
the projecting quantities is equidistant from said reactive layer,
causing relative movement between said holder and said plate while
maintaining said parallel relationship for disposing them closely
adjacent each other whereby said projecting quantities
simultaneously contact said reactive layer to simultaneously
commence reactions between said solutions and said reactive layer,
and separating said holder from said plate whereby substantially
equal portions of said projecting quantities transfer to said
reactive layer for completing said reactions.
2. A method as set forth in claim 1 wherein an inhibitor is applied
to said transferred quantities to simultaneously terminate said
reactions whereby said reactions are made substantially concurrent
with each other.
3. A method as set forth in claim 1 wherein said quantities include
standard solutions to which the other reactions are compared and
evaluated.
4. A method as set forth in claim 1 wherein a record of said
reactions is preserved upon said reactive substrate.
5. A method as set forth in claim 1 wherein said portions of said
quantities are caused to project below said holder by suspension of
said portions below said holder.
6. A method as set forth in claim 1 wherein said portions of said
quantities are caused to project from said holder by causing a
meniscus to project above said holder.
7. An apparatus for simultaneously testing the reaction of a number
of solutions with a reactive layer comprising a solution-holding
plate having a number of equal cavities which are constructed and
arranged for independently holding and supporting substantially
equal quantities of said solutions projecting substantially equal
distances from said solution-holding plate, a reaction plate having
portions disposed parallel to and equidistantly from corresponding
portions of said solution-holding plate, said reaction plate having
supporting means for holding said reactive layer, parallel-acting
movable coupling means connecting said solution-holding and
reaction plates whereby the relative distances between said
parallel plates may be parallelly varied from substantially
separated to closely adjacent positions, said cavities having a
shape and size for retaining said quantities of solution with a
portion thereof projecting at said substantially equal distances
therefrom whereby said projecting portions simultaneously contact
said reactive layer on said reaction plate when said plates are
moved into said closely adjacent positions and for substantially
transferring equal quantities of said solutions to said reactive
layer on said reaction plate upon subsequent separation of said
plates.
8. An apparatus as set forth in claim 7 wherein said cavities
comprise holes through said plate, said projecting portions of said
quantities being suspended from said holes below said plate, and
said reaction plate being disposed below said solution holding
plate.
9. An apparatus as set forth in claim 7 wherein said movable
coupling means includes a holding element whereby said solution
holding plate is maintained in a separated position from said
reaction plate.
10. An apparatus as set forth in claim 7 wherein said movable
coupling means includes adjustable stops for varying the distance
between said projecting quantities and said reaction plate in said
closely adjacent position of said plates.
11. An apparatus as set forth in claim 7 wherein said cavities are
substantially cylindrical.
12. An apparatus as set forth in claim 11 wherein said cavities
have a width of several millimeters.
13. An apparatus as set forth in claim 7 wherein said movable
coupling means comprises rod and slide bearing means.
14. An apparatus as set forth in claim 13 wherein said
solution-holding plate comprises a frame plate and a perforated
insert plate, said perforated insert plate being removably mounted
in said frame plate.
15. An apparatus as set forth in claim 7 wherein a removable sheet
is disposed on said reaction plate for contact with said drops, and
a framed holder being mounted upon said reaction plate for
receiving and holding said sheet flat upon said reaction plate.
16. An apparatus as set forth in claim 15 wherein said sheet
comprises photographic film, and a film container facing mounted
upon said reaction plate for supplying said film to said
holder.
17. An apparatus as set forth in claim 7 wherein said cavities
comprise wells in said plate, said projecting portions of said
quantities being menisci of said drops projecting above said
drop-holding plate, and said reaction plate being disposed above
said solution holding plate.
18. An apparatus as set forth in claim 17 wherein said solution
holding plate comprises a shallow tray incorporating a number of
said wells.
19. An apparatus as set forth in claim 18 wherein said tray and
said movable coupling means include tongue and groove means for
removable engagement with each other.
20. An apparatus as set forth in claim 17 wherein said movable
coupling means comprises a press having a base and a movable
plunger disposed above said base, said base having receiving means
for insertion of said solution holding plate, said plunger having
removable attaching means for engagement by said reaction plate,
and stop means reacting between said plunger and said base for
terminating the movement of said plunger toward said reaction
plate.
21. An apparatus as set forth in claim 20 wherein said layer has a
platen-supporting plate attached to said plunger, a platen
removably engaged with said platen-supporting plate, said platen
including a frame, and detachable means removably connecting said
platen with said frame.
22. An apparatus as set forth in claim 21 wherein said detachable
means comprises slots in said frame and engaging edges of said
platen-supporting plate.
23. An apparatus as set forth in claim 21 wherein said platen and
said solution plate have portions projecting a short distance
outwardly from said base to facilitate insertion and removal of
said platen and said solution holding plate.
24. An apparatus as set forth in claim 21 in which a transparent
coordinate plate is attached to the lower surface of said
frame.
25. An apparatus as set forth in claim 24 wherein said transparent
plate comprises a glass plate.
26. An apparatus as set forth in claim 24 wherein a slot is
disposed on said reaction plate between said film container and
said film holder to facilitate the severing of portions of said
film in said holder from said container.
Description
BACKGROUND OF THE INVENTION
This invention relates to a method and device for simultaneously
testing a number of different solutions in a predetermined period
of time. Separate timed tests for each solution require painstaking
care, considerable laboratory time and still incur the risk of
human error. An object of this invention is to provide a simple and
economical method and apparatus for testing a number of solutions.
Another object is to provide such a method and apparatus for tests
which must be of uniform duration.
SUMMARY OF THE INVENTION
In accordance with this invention, a plate having cavities such as
a perforated upper plate carrying drops of solution suspended from
its holes is moved closely adjacent to a lower plate carrying a
reactive substrate. The plate with cavities may also be a lower
plate with a number of wells in an upper surface into which an
excess of solution is deposited to make the drops bulge up above
it. The welled plate is made of a material which is not wet by the
solution (hydrophobic) to prevent the drops from spreading out of
the wells. An upper reactive layer is moved down into contact with
the bulging drops on the welled plate. This simultaneously contacts
the drops with the reactive layer and partially transfers an equal
volume of each to the reactive layer when the plates are separated.
All of the test reactions thus simultaneously commence and can be
simultaneously terminated by application of a suitable inhibitor.
The upper and lower plates may be movably connected by rods and
slide bearings or by a single plunger. Film storage and a holding
frame may be conveniently mounted on the lower plate below a
perforated upper plate. The welled lower plate may be removably
inserted below a reactively surfaced plate which is removably
attached to a plunger.
BRIEF DESCRIPTION OF THE DRAWINGS
Novel features and advantages of the present invention will become
apparent to one skilled in the art from a reading of the following
description in conjunction with the accompanying drawings wherein
similar reference characters refer to similar parts in which:
FIG. 1 is partially exploded pictorial view of one embodiment of
this invention;
FIG. 2 is a front view in elevation of the embodiment shown in FIG.
1;
FIGS. 3-6 are schematic side views in elevation of a simplified
form of the device shown in FIGS. 1 and 2 in successive phases of
operation;
FIG. 7 is a front view in elevation of a further embodiment of this
invention in a preliminary phase of operation;
FIG. 8 is a side view in elevation of the embodiment shown in FIG.
7;
FIG. 9 is a top plan view in elevation of the embodiment shown in
FIGS. 7 and 8;
FIG. 10 is a bottom plan view in elevation of the embodiment shown
in FIGS. 7-9;
FIG. 11 is a top plan view of the drop-holding plate utilized in
the embodiment shown in FIGS. 7-10;
FIG. 12 is a bottom plan view of the composite platen utilized in
the embodiment shown in FIGS. 7-10;
FIG. 13 is a cross-sectional view taken through FIG. 11 along the
line 13--13, and in the process of being filled with drops of
solution;
FIG. 14 is a cross-sectional view taken through FIG. 9 along the
line 14--14, but in the downwardly compressed position;
FIG. 15 is a front view in elevation similar to FIG. 7 but in the
upwardly returned position subsequent to that shown in FIG. 14;
and
FIG. 16 is a cross-sectional view taken through FIG. 7 along the
line 16--16 but in a slightly downwardly compressed position before
contact between the lower surface of the platen and the drops held
in the lower plate.
DESCRIPTION OF THE PREFERRED EMBODIMENTS (FIGS. 1-3)
In FIG. 1 is shown an apparatus 10 for simultaneously testing the
reaction of a number of different solutions with a common reactive
substrate. Apparatus 10 includes an upper plate 12 movably mounted
relative to lower base plate 14 by coupling 16 including four rods
18 sliding within collar bearings 20. Upper plate 12 includes a
perforated holder 22 removably mounted within recess 24 in frame
plate 26. Positioning pins 28 extend through corresponding holes 30
in perforated holder 22. Perforated holder 22 includes a number of
right cylindrical wells 32.
The shape of holder 22, its thickness and material are designed to
have an adhesive force for drops 34 of liquid solution to hold a
sufficient volume of liquid within and a sufficient distance below
wells 32 to contact lower reactive substrate 38, as later described
in detail. Upper holder 22 has an array of holes or wells 32
machined through it (for example right cylindrical) and is made of
a material which has lower adhesive forces for some liquid than
does the material of lower reactive substrate 38 for the same
liquid. If the liquid is water, for example, upper plate 12 could
be composed of a plastic and lower reactive substrate of a gelatin
film. Other useful shapes for wells 32 are, for example, right
elliptical or other right free-form shapes.
A certain volume of liquid is deposited (using a pipette or other
convenient volume delivery system) into each well 32 of upper plate
12 forming the suspended drop configuration 36 shown in FIG. 2. The
volume of the liquid required to form a stable (with respect to
mechanical disturbance) suspended drop primarily depends upon the
density of the liquid, its surface tension, the magnitude of the
adhesive forces between the liquid and the surface material of the
plate per unit of contact area, and the size and shape of the hole.
For example, an extremely stable drop is formed when 50 .times.
10.sup..sup.-6 liters of water are deposited into right cylindrical
holes of 3.5 millimeter diameter in a 4 millimeter thick acrylic
resin plate as shown in the drawings.
Lower base plate 14 includes upper surface 40 upon which common
reactive substrate 38 is disposed for contact by suspended portions
36 of drops 34. Substrate 38 is, for example, the gelatin surface
of an unexposed but developed sheet of photographic film, which is
useful in the study of trypsin-catalyzed reactions in which the
gelatin substrate emulsion, is hydrolyzed by different
concentrations of aqueous trypsin solutions, to form peptide
products.
Film 38 is conveniently stored within container 42 mounted at one
side of base plate 14 and is maintained in a flat position within
hinged framed holder 44. End 46 of film 38 is conveniently retained
within rectangular wire loop 48 at the end of base plate 14 remote
from film container 42. Notch 50 in upper surface 40 of base plate
14, between film container 42 and holder frame 44, provides a
convenient means for severing used pieces of film 38 from container
42. Guide wire 52 aligns passage of film 38 through holder frame
44.
Frictional disc brake 54 attached to one corner of upper plate 12
and in frictional contact with adjacent rod 18 gently holds upper
plate 12 in position, and its holding force is easily manually
overcome when upper plate 12 is moved throughout its range of
operative positions, including the lower position against
adjustable stop collars 56. Frictional disc brake 54 is made, for
example, of suitable frictional material such as a resilient
compressible washer made of felt or sponge material (synthetic
sponge rubber or polyurethane sponge).
Apparatus 10 is useful for conducting a number of simultaneous
tests of unknown solutions. It is particularly useful where the
reactions of a number of unknown solutions must all be compared
with the reaction of standard solutions with the same reactive
substrate in a uniform period of time. This device as previously
described is particularly useful in determining the trypsin
inhibiting capacity of human sera by comparing them side by side
with simulated normal, heterozygous and homozygous antitrypsin
sera.
Such tests are described in detail in U.S. Pat. No. 3,730,843.
Performance of the test described in the aforementioned copending
application by conventional methods would require considerable time
and care. The method and apparatus of this invention permits a
great number of such tests to be simultaneously and accurately
conducted.
OPERATION (FIGS. 3-6)
FIGS. 3-6 illustrate the performance of the method of this
invention by a simplified apparatus 10A shown in FIG. 3 ready for
use. A number of trypsin solutions of 50 .times. 10.sup..sup.-6
liters are deposited with an automatic pipette into holes or wells
32A of upper plate 12A shown in FIG. 4. Upper plate or carriage 12A
is lowered manually until contact with stops 56A is made as shown
in FIG. 5.
When contact is made, the liquid samples simultaneously make
contact with film 38A as shown in FIG. 5. The distance between
plates 12A and 14A at contact, governed by the size or position of
stops 56A, is one of the determining factors of the magnitude of
the contact area of the liquid with film 38A on lower plate 14A. At
contact, a timer (not shown) is activated either manually or by
electrical contact between upper plate 12A and stop collar(s)
56A.
Following contact upper plate 12A is returned to its original
position thereby, effecting the partial transfer of an equal volume
of each liquid sample in drops 37A to film 38A as depicted in FIG.
6. The efficiency of the transfer can be maximized for any given
system and configuration if the reactive substrate-liquid forces
are greater than the upper plate-liquid forces. When, for example,
50 .times. 10.sup..sup.-6 liters of water are deposited in the
right cylindrical holes of 3.5 millimeter diameter, approximately
20 percent of the water is transferred from the upper acrylic plate
to a gelatin film reactive substrate when the stops 56A are set so
that a 1 millimeter gap exists between the plates at contact. In
contrast, if the gelatin film reactive substrate is replaced with
polystyrene, only 10 percent of the water in holes 32A is
transferred to the polystyrene surface.
At some designated time (t = x) lower plate or carriage 14A
containing film 38A and its array of about 10 .times.
10.sup..sup.-6 liter drops 37A of reaction mixture is transferred
to a low pH (2-4 pH) bath (not shown). Upon immersion of film 38A,
the catalytic action of the trypsin is terminated and the
hydrolyzed gelatin is removed. The extent of hydrolysis of the
gelatin is then assessed by transmission of light through the
spotted areas of film 38A. The use of a color film 38A, as
described in the aforementioned copending patent application,
provides a tricolored gelatin sandwich in which the depth of
reaction penetrating is read with remarkable facility.
(FIGS. 7-16)
In FIGS. 7-16 is shown another testing apparatus 10B which
primarily differs from apparatus 10 and 10A in that solution drops
34B are contained within bottomed wells 32B instead of open holes
or wells 32 and 32A. Plate 12B further differs in that it is the
lower plate from which projecting portions 36B of drops 34B project
upwardly above plate 12B instead of being suspended below as shown
in FIGS. 1-6. There are also other corresponding differences as
described in the following.
In FIGS. 7 and 8 drop-holding plate or tray 12B is inserted within
chamber 60B of press apparatus 10B having a composite platen 62B
secured to plunger 64B by screws 66B. Cap 68B is mounted over and
secured to the top of plunger 64B and its includes a tubular skirt
70B inserted within sleeve bearing 72B at the top of press housing
60B. Compression spring 74B reacts between the bottom of tubular
skirt 70B and the top of housing 60B to cause cap 68B and plunger
64B to return upwardly after a downward stroke. Removable plate 12B
or tray is inserted above base 76B of housing 60B with its flanges
78B and 80B inserted under the bottom edges of retaining pins 82B
disposed within housing 60B in a tongue and groove-like manner.
FIGS. 11, 12 and 13 show details of removable welled plate 12B
including a number of drop-holding bottomed wells 32B holding drops
34B of solution. FIG. 13 shows wells 32B being filled with
predetermined excess amounts of solution from pipette 84 sufficient
to cause upper projecting portions 36B of solution to project above
the upper surface 13B of plate 12B to cause contact with composite
platen 62B and the reaction layer 38B on it later described. The
material of which plate 12B is made of a type which will not be wet
by the contained solution (hydrophobic) therefore preventing the
solution from spreading over the surface of the plate and causing
it to form a convex meniscus comprising projecting portion 36B
shown in FIG. 13. The result achieved is that for each liquid
sample placed in a well there occurs a convex meniscus of the
liquid of a given height above the plate surface. For example, when
each 3/16 inch diameter, 3/16 inch deep well of the present
injection-molded polystyrene plate is filled with 0.10 milliliters
of liquid sample, there results a convex meniscus the maximum
height of which is 1.2 mm above the upper surface of the plate. The
reason for the preference of this type of plate as opposed to the
plate containing the perforated holes from which the liquid samples
are suspended is one of convenience. It has been found that less
skill and time is required to deliver the sample to a well than
into a hole and, more importantly, that the well principle
eliminates the possibility of the sample falling through the plate
due to a too rapid release of the liquid sample from the
pipette.
Plate 12B is for example injection molded of polystyrene to provide
wells 32B in its upper surface and flanges 78B and 80B. Flange 78B
as shown in FIG. 11 extends the entire length of plate 12B and 80B
extends less than the full length to index plate 12B for insertion
in only one position into press housing 60B in conjunction with
stop 86B (FIG. 10). FIG. 11 also shows identifying coordinates A-E
and 1-5 for wells 32B and also for the two control drop-containing
holes 32B designated HET and HOM.
FIG. 12 shows the bottom of the removable elements of platen 62B
including a coordinated glass plate 88B removably adhered, for
example by rubber cement, to the bottom of platen frame 90B.
Coordinated glass 88B includes ruled blocks 92B and indicia
corresponding to positions of wells 32B in lower plate 12B. Glass
plate 88B also includes an extending tab 94B to provide a handle
for the removable assembly shown in FIG. 12. Tab 94B may
accordingly be roughened to facilitate grasping and application of
identifying marks.
The transparent film-holding plate 88B is silk-screen coded on its
upper surface (non-film surface). The coding design matches the
particular array of samples on the film as dictated by the spacing
of wells on the well plate. In the particular case of the
alpha-1-antitrypsin mass screening test, this coding facilitates
the rapid identification of any positive reaction when light is
passed through the normally opaque dye/gelatin film substrate and
further facilitates the comparison of a positive sample spot with
the two reference spots labeled HET (heterozygote deficiency) and
HOM (homozygote deficiency).
FIG. 7 shows assembled frame 90B and glass plate 88 B inserted in
housing 60B with lateral slots 96B of frame 90B engaged about the
edges of platen-supporting plate 98B, which is secured to the
bottom of plunger 64B by screws 66B. In FIGS. 7, 14-16 is also
shown reaction layer 38B of unexposed but developed color film as
previously described. Reaction layer 38B is adhered to the bottom
of glass plate 88B by a suitable adhesive, such as rubber
cement.
OPERATION (FIGS. 7-16)
Drops of different solutions are placed in wells 32B by pipette 84
as shown in FIG. 13 to cause an excess of solution to raise
projecting menisci 36B of solution above upper surface 13B of plate
12B. Plate 13B is then inserted within the housing 60B of base 76B
as shown in FIGS. 7-10. A piece of unexposed but developed color
film 38B corresponding in area to the coordinated portion of glass
plate 88B is then attached to the bottom of glass plate 88B by a
liquid adhesive, such as rubber cement or for example a
double-sided adhesive transparent tape. Reaction layer or film 38B
is mounted or bonded to glass plate 88B on the unreactive side of
the film, that is by its celluloid or other backing which is bound
to the glass by means of an adhesive.
A composite platen 62B is then prepared as shown in FIG. 12 by
adhering glass plate 88B to the bottom of frame 90B which is then
removably connected to plunger 64B by engaging grooves 96B about
platen supporting plate 98B. Platen 62B is then moved toward
projecting drop menisci 36B, as shown in FIG. 16 and ultimately in
FIG. 14 to cause reactive layer 38B to contact projecting menisci
36B of drops 34B. As a result of downward pressure in the direction
of arrow 100B on plunger cap 68B, the downward motion of plunger
64B is arrested by contact of shoulder stop 102B of cap skirt 70B
with the upper periphery 104B of housing sleeve bearing 72B. Stop
surfaces 102B and 104B are predetermined to cause menisci 36B to
simultaneously touch the surface of film 38B a predetermined amount
sufficient to cause the transfer of drop portions 37B shown in FIG.
15 of sufficient volume upon upward movement of plunger 64B in the
direction of arrow 106B to initiate substantial reactions for the
particular comparison test being conducted. Return movement of
plunger 64B is accomplished by releasing cap 68B which is
automatically raised by return expansion of compression spring 74B.
The size of the transferred drops 37B may be adjusted by changing
the dimensions of surface 102B and 104B by adjusting configurations
or inserting stop washers (not shown).
Film 38B containing transferred drops 37B may be left in position
in housing 60B until the reaction time is completed or may be
removed together with glass plate 88B and frame 90B (shown in FIG.
12) and stored until the end of the reaction time. Since film 38B
is hydrophilic it can be stored upright or on its side without any
danger of drops 37B running together. After the reaction time is
completed and film 38B immersed to terminate the reaction, the
extent of hydrolysis of the gelatin for each sample may be read
from the coordinated assembly shown in FIG. 12 together with the
adhered film layer 38B. Drop-containing plate 12B is removed from
housing 60B and discarded after transfer of projecting menisci 36B
of drops 34B.
As one skilled in the art can readily appreciate, the plate 88B can
be composed of a wide variety of materials other than glass,
including plastics, metals or wood. Further, plate 88B can be solid
or may comprise a rigid frame to which the film 38B is adhered.
* * * * *