CA2061984A1 - Capillary stop-flow junction having improved stability against accidental fluid flow - Google Patents

Abstract

CAPILLARY STOP-FLOW JUNCTION
HAVING IMPROVED STABILITY AGAINST ACCIDENTAL FLUID FLOW
ABSTRACT OF THE DISCLOSURE
A capillary stop flow junction located in a housing at an end of a capillary passageway used to transport a liquid and at the beginning of a non-capillary internal chamber in the housing, in which the stop-flow junction contains an improvement which comprises 1) means for selectively trapping a gas in the capillary passageway and non-capillary chamber, wherein when the means for trapping is activated and the liquid enters the capillary passageway, the gas is compressed by the liquid as the liquid flows through the capillary channel and stops flowing at the stop-flow junction; or 2) a stop-flow nozzle surrounding the capillary passageway and projecting into the chamber; or 3) the stop-flow junction being formed from a single housing body member; or 4) a rupture junction in the capillary pathway, wherein the rupture junction is a stop-flow junction providing less maximum available back pressure than the capillary stop-flow junction. Diluters capable of serial dilution that use the stop-flow junctions of the invention are also described.

030191 67.

Description

BIOT-033/OOUS ~9 8 ~

CAPILhARY STOP-PLOW JUNCTION
~L~ING IMPROVED STABILITY AGAINST ACCIDENTAL FLUID FLOW

INTRODUCTION

Technical Field This invention relates to methods and appara-tuses used for controlled transport of liquids by capillary action and gravity, particularly the automakic measuring and diluting of small volumes of liquids using cartridges in which flow of sample and diluent is controlled at a junction between capillary-flow and non-capillary-flow regions, referred to herein as a stop-flow ~unction.

Back~

The phrase "stop-flow junction~ was intro~uced to describe a control region in a capillary passageway that is used in a number of prior inventions arising out of the laboratories of the present inventors. A stop-flow junction is a region in a fluid track that marks the junction betwee~ an early part of the fluid track in which sample flows by capillary action (and optionally gravity) and a later part of the fluid track into which sample does not normally flow until flow is initiated by : some outside force, such as an action of the user.
A stop-flow junction is not a traditional : val~e as it has no moving parts. Rather, this junction relies on back pr~ssure from ~he surface tension of the liquid sample to stop flow. This back pressure can be created in a number of ways. ~or example, back pressure is created when the cross-sectional area of a liquid flowpath increases in a region in which there is contact between the liquid and the container walls (e.g., when a 030191 1.

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2~19g4 small tube enters a larger chamber or when the cros~
sectional area of a channel increases). More consistenk operation of a stop-flow junction is achieved when the increase in cross-sectional area of the flowpath is abrupt rather than gradual, particularly when there is a break in capillarity in the sample flowpath. In many cases, the ~unction will be formed when a small-diameter capillary channel enters a larger, non-capillary chamber. A small channel or tllbe can enter the larger chamber at a right angle or at an angle other than a right angle. The angle between the internal wall of the small tube and the surface of the chamber in the latter case will be different at different locations around the circumference of the junction.
In general, for small (capillary-size) junc-tions, the back pressure will be largely determined by the smallest radius of curvature assumed by the meniscus. For example, when a capillary tube with a circular crosssection enters a larger space so that liquid bulges out into the ~pace under hydrostatic pressure, the meniscus will be approximately spherical, and the back pressure (~p) is gi~en by the Young-~aplace equation: ~p = 27/R, were ~ is the surface tension of the sample fluid and ~ i5 the radius of curva~ure. See, Miller and Neogi, ~Interfacial Phenomena. Equilibrium and Dynamic Effects", Marcel Dekker, Inc., New York, 1985, and Davies and Riedeal "Interfacial Phenomena", 2nd Ed., Academic Press, New York, 1963. If the fluid meets the surface at an angle greater than 0, this back pressure will be reduced by a geometric term. The radius, R, will change ( become smaller ) as the hydrostatic pressure increases, so that the back pressure and hydrostatic pressure balance. As hydrostatic pressure increases, R reaches a minimum value (maximum curvature~
determined by the geometry of the device and the contact angle. The corresponding back pressure defines the 030191 2.

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maximum hydrostatic pressure sustainable by the stop-flow junction.
Back pressure i~ also created when the surface that the liquid contacts changes to decrease adhesion between the liquid and the container wall ( for example, when an aqueous sample moves from a hydrophilic to a hydrophobic surface). The surface properties of the various interior surfaces of the device of the invention can and generally will be controlled by various physical and/or chemical treatments. For a discussion of controlling surface propertie~ o~ similar devices, see commonly assigned U.S. Applicalion Serial No. 8~0,793, filed July 1, 1986. For examp:Le, plastic surface~ can be treated to increase their hydrophilicity. Either the whole apparatus or specific parts can be treated.
Alternatively, different parts of the apparatus can be made o~ different plastics. For capillary flow, contact angles of les~ than 90 are sufficient, preerably 10-85 and most preferably 30-60. In order to provide these contact angles for aqueous samples, the capillary surfaces will be hydrophilic (at least to some measurable extent). For non-a~ueous li~uids, a hydrophobic surface would be appropriate. By u~ing a combination of container wall geometry and surface wetability, a back pressure range of from 0 (no change in cross-sectional area or surface adhesion) to 20 cm H20 and higher can easily be achieved with water as the liquid. When the back pressure i5 0, the location in question is not a stop-flow junctionO A stop-flow junction occurs when there is sufficient back pressure to prevent the flow of sample past a particular point in the flowpath; e.g., from the measuring chamber to the receiving chamber of a dilution apparatus as described herein.
When considering the amount of available back pressure for any given design, the realities of manufacturin~ and of the physical world at the microscopic level must be considered. Imperfections in 030191 3.

.~ , . ' . ' ~ ' . ' , , the container walls duriny gradual widening of chambers may cause liquid to "creep~ more on one side than another, thereby allowing the stop-flow junction to fail. Liquid can al~o creep around corners when imperfections are present that result in unbalanced forces. Unbalanced forces will also be present when the junction is not horizontal. A horizontal junction, for example, occurs when a vertical tube enters the top horizontal surface of a chamber. If a horizontal tube enters a vertical wall of a container, a vertical junction is present, and the pre~sure at the bottom of the stop-flow junction will be greater than the pressure at the top of the ~unc~ion, due to hydrostatic pressure caused by the different height~ o~ liquid. Nonetheless, non-horizontal stop-flow ~unction~ can be cxeated by reducing the diameter of the smaller channel containing li~uid a~ it enters the larger area, thereby reducing the difference in pressure between the upper and lower portions of the ~unction, and other manufacturing imperfection~ can be alleviated by quality control operations, although wi~h increased costs of manufacturing.
; U.S. Patent No. 4,426,451, which waY developed in other laboratories, describe~ a number of regions that it refers to as "meniscus control means" for u~e in a device in which there i~ capill ry flow from one capillary zone to another. The meniscus control means described in that pa~ent can be used in apparatuses in which capillary/capillary tran~itions and temporary ;~ 30 stoppage of flow is desired before flow continues into the next zone. However, the patent is not directed to stopping flow when the ~econd zone is not a capillary zone. In contrast to the specific teachings of the '451 patent, which indicate that the walls of the capillary chamber mu~t gradually narrow and gradually e~pand in order ~o provide for flow stop, an abrupt widening has been found to be more effective in the practice of the present invention when the second cham-030191 4.

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2 ~ 8 4 ber is not a capillary space. Although it is recognized that imperfections will exist on the molecular level, it is preferred that t:he ~unction be as sharp as possible from a macroscopic view point, approaching ag closely as possi.ble the ideal junction formed by the inter~ection of the surface (which can be curved) forming the walls of the measuring chamber with the surface forming the wall of the receiving chamber surface in which the stop-flow junction is found (which can also be curved~. Maintaining a horizontal ~unction to avoid pressure differentials, reducing the area of the junction, changing the surface of the capillary so as to decrease the hydrophilic character (for aqueous solutions), providing smooth surfaces (rough Rurfaces encourage creep of liquid along the surface), and pro-viding an abrupt change in cross-~ectional area (pre-ferably providing an angle between intarsecting ~urfaces of about 90 or lower) all operate to prevent creep of liquid from one chamber to the other.
It should be recognized that flow stop can oc-cur both stably and metastably. A metaRtable flow stop is one in which flow stops on the macroscopic level but may resume without apparent cause after a time interval of a few seconds to a few minutes. Gradual creep of liquids along container walls or through microscopic or submicroscopic channels resulting from imperfections in the manufacturing proce~ is believed to be the mechan ism by which flow starts again once i has stopped.
~dditionally, vibrations (such as might be caused by persons walking near the apparatus or starting and stopping of nearby equipment, such as air-conditioning units) may also be sufficient to start flow in a metastable situation~ However, there is no requirement of absolute stability in cases where an apparatus is designed for addition of a diluent and eventual starting of flow at the stop-flow ~unction. Accordingly, any flow stop which can be sustained for at least 10 seconds, preferably at least one minute, and more 03~1~1 5.

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preferably at least five minutes, i9 sufficient fo ~ ~19 in a diluter.
Although these prior stop-flow junctions were sufficient for most uses, improvements in skability of the stop-flow junction against accidental start has been desirable from the point of view of developing a commercial apparatus. A numbe:r of factors contribute to the instability of the ~unction. For example, variations in the sample physical properties (such as density, viscosity, hematocrit, microheterogeneity, surace tension, and contact angle with housing walls) can affect both the forward pressure acting to ~avor flow and the back pressure available at the stop-flow junction to stop flow. Density controls the hydrostatic pressure at the ~unction. Surface tension and contact angle determine the pressure that the junction can exert in opposition to ~low. Viscosity determines the rate at which the sample moves to the ~unction and therefore the excess back pressure (over that nece3sary for an equilibrium state) required to prevent the momentum of the sample from breaking through the junction.
Hematocrit of blood sample affects both viscosity and ~,/
density. Microheterogeneity has an impact on local properties at the junction, which can vary significantly from the bulk properties of the sample. Other vaxiations include sample volume, which affects hydrostatic pressure by varying the height of the upper sample surface above the junction; method of sample application by different uses (or the same user ak different times); variations from lot to lot of the physical properties, such as contact angle with a standard liquid, of the housing out of which the diluter is made; variations in the size and ~hape of the junction arising during manufacturing, such as can be caused by plastic "burrs" at corners and edges; and local external actors, such as mechanical vibrations caused by nearby machinery or foot travel, as well as 030191 6.

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variations in orientation of the diluter from a horizontal operating position.
While it is possible for any of the previous diluters arising out of the inventors' laboratory to be used despite these potential problems, such as by designing a monitor in which the diluter will be used that is capable of detecting when flow accidentally starts prior to the desired time, improvement of the reliability of operation is highly desirable. For example, few patients desire having a second finger puncture for the purpose of obtaining a second blood sample. In other cases, the patient may have left and no more sample may be available, thereby inconveniencing both the patient and the physician. Thu8, there remain~
a need for ~mproved stop-flow ~unctions having increa~ed stability against accidantal fluid flow and or diluters that incorporate these improved features.

Relevant Literature .
West German published patent application DE3328964Cl, publication date February 14, 1985, de-scribes a device for the automatic, discontinuous sam-pling of fluids using a capillary tube that acts as a measuring device and which can be either dipped into a fluid being sampled or alternatively moved into a posi-tion from which the sample is transported with a diluent to an analyzer by a pump or suction. U.S. Patent No.
4,454,235 describes a capillary tube holder for liquid transfer in immunoassays. U.S. Patent No. 4,233,029 describes a liquid transport device formed by opposed surfaces spaced apart a distance effective to provide capillary flow of liquid without providing any means to control the rate of capillary flow. U.S. Patent Nos.
4,618,476 and 4,233,029 describe a similar capillary transport device having speed and meniscus control means. U.S. Patent No. 4,426,451 describes another ~ ~ similar capi:Llary transport device including means for ?

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stopping flow between two zones, flow bein~ resumed by the application of an externally-generated pressure.
U.S. Patent Nos. 3,811,326; 3,992,150; 4,537,747; and 4,596,780 describe various processes and devices in which a capillary tube is u~ed to take up a predetermined volume of the test solution and the charged capillary is then placed in a cuvette or other container of liquid that is used as reagent or diluent.
U.S. Patent No. 3,799,742 desc:ribes an apparatus in which a change in surface character from hydrophilic to hydrophobic is used to stop flow of a small ~ample, thereby metering the sample present. U.S. application serial number 117,791, filed November 5, 1987, and U.S.
application serial number 090,026, filed August 27, 1987, both of which are assigned to the same as~ignee as the present application, described a number of dilution and mixing cartridges.

SVMMARY OF THE INVENTION
The present invention provides an improved stop-flow junction for use in, among other potential locations, a self-contained dilution apparatus that does not require the use of externally generated force (except gravity) to move liquids between its various part~ or to provide for reproducible dilution o~
samples. The principal motive force in such devices arises from capillarity and gravity (resulting in hydrostatic pressure), thus giving rise to the name stop-flow junction, since a stop-flow junction occurs at the junction of a capillary region and a region where flow does not occur solely as a result of capillarity and gravity.
Stop-flow junctions are described herein that provide increased stability in the "stop" state. A
series of individual improvements are available in accordance with the present invention, or all o~ t~e improvements can be present in the same device.

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Specifically, tho dovice o tha invent.ion comprises a capillary stop-flow junction located in a housing at ~n end of a capillary passageway Por tran~porting a liquid and at the beginning of a non-capillary chamber, in which an improvement is present which comprises:
a. means for selectively trapping a gas in said capillary passageway and non-capillary chamber, wherein when said means for trapping is activated and said liquid enters said capillary passageway/ said gas is compressed by said liquid as sa:Ld liquid flows through said capillary channel and stops flowing at said stop-flow junction; or b. a stop-flow nozæle surrounding said capillary passageway and pro~ecting into said chamber;
c. a stop ~low junction formed from a single housing body member; or d. a rupture ~unction in said capillary pathway, wherein said rupture junction is a stop-flow junction providing less bAck pressure than said capillary stop-flow junctionO
One, some, or all of thess improvements can be present in a single stop-flow junction of the invention.
The improved stop-flow ~unctions of the inven~ion can be used in a diluter that, in addition to containing the improved ~top-flow ~unctions, also provides other advantages bscause of its improved design, such as improvements in reproducibility of sample measurement and dilution control. The improved diluter is an apparatus for automatically carrying out a dilution of an aqueous sample with one or more a~ueous diluents in a housing, comprising in said housing:
(1) a sample application site for receiving a sample;
(2) a rupture chamber comprising a ~ented interior chamber;

(3) a mi.xing chamber comprising a vented interior chamber having a first volume;

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(4) a diluent application site for receiving ~ 0619 8 diluent;
(S) capillary flow means comprising:
(a) a central valved segment having a first and a second end;
(b) a ~alve located i.n said central valved segment;
(c) a sample segment connecting said sample application site to said first end of said central valved seyment;
(d) a rupture segment: connecting said rupture chamber to said ~irst end of said central valved segment; and (e) a measuring segment connected to said lS second end o~ said central valved segment and having first and second exits, wherein said first exit connect~ said measuring segment to said mixlng chamber and wherein said measuring segment has a second volume ~maller than said first volume of said mixing chamber;
(f) a first ~top-flow junction located at said first exit of said measuring segment and adapted to the surface-tension characteristics of the sample so as to provide sufficient back prsssure resulting : 25 from contact between the sample and wall means of said housing at said first stop-flow ~unction to prevent sample from flowing through said first stop-flow junction in the absence of diluent;
(g) a second-stop flow junction located at : : 30 said second exit of said measuring segment and adapted to the surface-~ension characteristics of the sample so as to provide sufficient back pressure resulting from contact between the sample and wall means of said housing at said second stop flow junction to prevent sample from flowing through said second stop-flow ~unction in the absence of diluent; and : 20299751 0301gl 10.

(h) a third stop-~low ~unction located ~ ot~ g 8 junct.ion of said rupture sec~ent and said rupture chamber and adapted to the surface--tension characteristics of the sample so as to provide sufficient back pressure xesulting from contact between said sample and wall means of said housing at said third stop flow junction to prevent sample from flowing through said th~rd stop-flow junction in the absence of diluent, wherein said third stop-flow junction provides less maximum-available back pressure than said first stop-flow ~unction;
whereby addition of sample to ~3aid sample application site causes sample to fill said capillary flow mean~;
and (6) diluent flow means connecting said diluent application site to said second exit o~ said measuring segment.

BRIEF DESCRIPTION OF T~IE DRAWINGS
The present invention will be better under-stood by reference to the following detailed description of the invention when considered in con~unction with the attached drawings that form a part of the present specification, wherein:
Figure 1 is a vertical cross-section of a first embodiment of the invention showing a vent-assisted stop-flow junction.
Figure 2 is a vertical cross-section of a second embodiment of the invention showing a stop-flow nozzle.
Figure 3A is a vertical cross-seckion of a prior~art stop-flow ~unction showing a stop-flow juntion formed at the ~unction of two separate housing members that have been welded together.
~igure 3B is a vertical cross-section taken along line B--B of the embodiment shown in 3A.
Figure 9 is a vertical cross-section of a 03~191 ll.

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further embodiment of the invention showing a through-body stop-flow junction of the invention.
Figure 5 is a vertical cross-section of still another embodiment of the invention showing a rupture junction in the capillary pathway that contains a stop-flow junction that is being stabilized.
Figure 6 is a vertical cross-section of a diluter of the invention showing a stop-flow junction having the principal features of the stop-flow ~unction embodiments of Figures 1, 2, 4, and 5 along with other features of the diluter as a whole.
Figures 7A throuyh 7,J are a series of vertical cross-sections of the embodiment o Figure 6 taken at locations A-A through J-J of the embodiment Figure 6.
Figure 8 in a schematic diagram of chemistry associated with a specific analy~is that can be carried out in the embodiment o~ Figures 6 and 7.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS
I. New stop-flow junction A. General background The present invention provides an improved stop-flow junction for use in apparatuses that require stoppage of capillary flow followed by controlled restart of flow. Such stop-flow junctions are particularly useful in apparatuses and methods in which small samples are automatically measured and diluted.
Such apparatuses are generally mall, convenient to use, and require no moving par~s for the movement of fluid, with gravity and capillary action being sufficient to provide all fluid motive forces required for the sample measurement and dilution steps. Such dilution and mixing cartridges are described in U.S. Patent No.
4,868,129, ll.S. Application Serial No. 117,791, filed November 5, 1987, and U.S. Application Serial No.

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337,286, filed April 13, 1989. However, the apparatuses of the present invention provide a number of improvements in stop-flow junctions relative to those described in previous dilution and mixing apparatuses, particularly in ease of manufacture and reliability of operation for large numbers of diluters made from the same mold. Among the specific improvements of the present apparatus are (1) means for selectively trapping a gas in a capillary passageway and non-capillary chamber adjacent to a stop-flow junction, wherein when said means for trapping is activated and a liquid enters said capillary pas~ageway, said gas i8 compressed by said liquid as said liquid flows through said capillary channel and ~tops flowing at said stop-flow junction;
t2) a stop-flow nozzle surrounding a capillary passageway and projecking into a chamber, with the stop-flow ~unc~ion being at the entrance of the capilary passageway into the chamber; (3) a stop-flow junction formed from a single housing body member; and (4) a rupture junction in a capillary pathway, wherein said rupture junction i a stop-flow junction providing less maximum available back pressure than said capillary stop-flow junction. Each of these improvements, which can occur alone or in combination with any other of these improvement~, is discussed in detail below.
The basic features of a stop-flow junction are described in the patents and patent applications identified above in the background section of this application. There are two required parts to a stop-flow ~unction, the first of which is a region in a fluidpathway in which fluid flow occurs either solely under the influence of capillary action or under the combined influence of capillary action and gravity. The junction exists at the end of this region of free flow at a transition to a region at which capillarity flow will cease, even in the presence of a gravitationally derived pressure arising from a liquid head above the capillary-stop junction. Well-known example~ of capillary 030191 13.

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junctions exist in familiar devices, such as a capillary tube used for obtaining blood samples from a finger puncture. In such a simple device, the stop-flow junction is the end of the capi].lary tube, since capillary forces xetain ~ample inside the tube, even when the tube is oriented vertically and gravitational forces are present on the sample. Other examples are described in the previously discussed publications and patent applications.
B. Vent-assisted stop-flow iunction ~ he first o~ the improvements that have been recognized and developed by the current inventors is a technique (and associated apparatuse~) Ln which a gas (usually air from the atmosphere surrounding khe apparatu~ in which the stop-flow junction i8 located) i~
trapped and compres~ed when a liquid enter~ the capillary portion of the passageway and flows through the passageway to the stop~flow junction. The trapping must be selective since the trapped gas will need to be vented in order for flow to continue unimpeded to other parts of the apparatus at an appropriate time. By properly selecting sizes of the compressed air space relative to the gravitational and c~pillary ~orces present in the device, reliability of flow s~oppage at the stop-flow ~unction can be increased many fold over.
Since the volume of the trapped gas is manipulated most easily by changin~ the size of the vent channels and chambers~ this aspect is referred to as a vent-assisted stop-flow junction.
The operation o~ a vent-assisted capillary stop-flow junction is readily understood by reference to Figure 1 and the mode of operation of the apparatus shown in the figure. However, it should be recognized that this is not the sole embodiment by which the present invention can operate and that the embodiment 030191 14.

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shown in Figure 1 is merely exemplary of thi~ aspect of the invention.
Figure 1 is a vertical, cross-~ectional schematic drawing of a dilution apparatu~ having a vent-assisted stop-flow junction. The diluter ~hown in Figure 1 is similar to the single-dilution apparatus described in U.S. Patent No. 4,868~129 with the additional flow directing chamber of U.S. Applicati.on Serial No. 07/337,286, filed April 13, 1989. R~ference may be made to this earlier patent and patent application for detail on the various parts of the apparatu~. The present discus3ion will address the vent-assisted stop-~low junction without prolonged discussion of other aspects of the device.
Cartridge 100 contains a sample application site 110, a capillary channel 120 leading from sample application site 110 to flow directing chamber 130, capillary measuring chamber 140, mixing chamber 150, capillary passageway 160 leading from flow directing chamber 130 to waste chamber 165, a rupturable container 175 of diluent in an internal chamber functioning as a diluent application site 170, and a channel 180 leading from the diluent application site to the flow directing chamber 130. All of these parts of the apparatus have been previously described in earlier patents and patent application4. Parts of the device relating specifically to the vent-assist feature include an initial capillary channel 101 leading to a relatively large interior chamber 102 referred to as a vent~surge chamber, capillary channel 103 connecting vent-surge chamber 102 to the environment surrounding cartridge 100, where vent opening 104 exists to allow atmospheric gases to enter and leave venting channel 103 and other interior chambers of ~he device, and vent closure 105, which is capable of being moved in the directions shown by the arrow to alternatively clo~e and open the vent at 104.
The operation of the vent-assisted stop-flow junction can readily be seen from the following 03~191 15.

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description and by reference to Figure 1. Prior to application o a ~ample to sample application site 110, vent closure 105 is moved to the left where it seals against the housing at vent 104. The vent clo~ure substantially seals the vent from the external environment. Any means that accomplishes this result is satisfactory, such as providing a flexible pad that presses against the surface of the housing at vent exit 104; providing a close-fitting, smooth disc that contacts a corresponding smooth surface on the houæing;
or any other effective means of sealing off the internal space in the housing from the surrounding atmosphere.
The vent closure is typically operated by a monitor into which the housing has been inserted.
After the vent i8 closed, sample is applied at sample application site 110. Sample flows through capillary 120 to flow directing chamber 130 and then into measuring chamber 140. When sample first enters measuring chamber 140, it create~ a sealed interior space consisting of measuring chamber 140, mixing chamber 150, and any Yenting spaces. In the embodiment shown in Figure 1, the venting spaces consist of capillary channels 101 and 103 and vent-surge tank 102.
However, this vent-surge tank i5 included merely ~o provide an appropriate volume for the trapped air or other gas present in the indicated chambers and i8 therefore optional. If measuring chamber 140, mixing chamber 150, and the vent spaces leading to vent exit 104 provide the desired compressible volume of air, no vent-surge chamber 102 is required. As sample flows down capillary measuring chamber 140, the air trapped in the enclosed space is compressed. This compressed air will act to oppose the forward motion of the liquid in the measuring chamber and ~hus act to ~tabilize stop-flow junction 145 at the intersection of measuringchamber 140 and mixing chamber 150.
Earlier applications from the laboratories of the present inventors have described vent closures that 030191 1~.

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were designed to stop flow of sample in capillary 2061 98 passageways without requiring the concurrent presence of a stop-flow ~unction. Such vent closures were different from those used in the present invention. The build~up of pressure in the enclosed space in the present invention is only sufficient to impede and partially counteract forward-directed pressure from the weight of the sample. If no stop-flow junction is present at a location where flow stoppage is desired and a vent closur~ is used in the manner Idescribed herein, forwaxd-directed pressure would cause ~ample to continue to flow beyond the desired location.
Since stop-flow ~unction 145 is designed so that flow will occur at this location during the dilution step, maximum capillary force available at this junction is designed to be weaker than the head pressure at the stop junction for all cases in which diluent is present and vent 104 i8 open. This can be achieved simply by selecting an appropriate size for the opening at stop-flow junction 145. When the opening at stop-flow junction 145 is circular, Formula 1 below allows design of an appropriate junction for any given sample and housing type merely by selecting an appropriate radius for the opening.
Formula 1:
dgh1 > 7 cos o d = density of sample g = gravitational constant hl = head on stop-flow junction (sample and diluen~) = surface tension of sample ~ = contact angle of sample on housing wall r = radius of opening at stop-flow junction It will be recognized that Formula 1 a~ove is intended for circular openings u~ed as stop-flow 20~99751 030191 17.

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junctions and that other shapes will require the use of different formulas. Other parameters that can be used to control flow are also evident from the formula. For example, the head height available can be adjusted by appropriate design of khe cart:ridge (e.g., a tall thin measuring chamber to maximize head height, or low-lying broad measuring chamber to min.imize height). The contact angle can likewise ~e used to control back pressure, either by selecting a material for manufacture of the housing (either the ent:ire housing or a part thereof) that provides the appropriate contact angle or by modifying the ~urface properties of the housing at an appropriate location, e.g., by plasma etching, as has been described in earlier patents, such as 4,756,884.
Empirical ad~ustment of head height and surface characteris~ic by appropriate design of the cartridge can be u~ed to control back pressure at a stop-~low junctions of any shape.
The maximum flow-opposing pressure created by compression of air in the internal spaces of the diluter when vent 104 is closed and a sample is applied should be equal to or less than the head precsure on the stop-flow junction. Equal internal pressure to balance the head pressure is preferred. This opposing pres~ure can be varied by varying the ratio of ~he pre-compressed and compres~ed volumes of air. In order to allow flexibility of design, vent-surge tank 102 can be provided in different volumes, since this part of the apparatus does not affect the dilution that occurs in mixing chamber 150 (an additional ~top-flow junction can be included in the early portion of the vent leading to the surge tank to keep the mixture from entering the surge tank). The volume of the surge chamber is selected so that the pre-compression and post comprecsion trapped-gas volumes are sufficient to satisfy the inequality set forth below in Fo~mula 2 below:

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Formula 2:
Vl dgh2 ~' P ( V - 1 ) d = density of sample g = gravitational constant h2 = head on stop-flow junction (sample alone) P = atmospheric pressure Vl = pre-compression trapped-gas volume V2 = post-compression trap]?ed-gas volume In the embodiment shown in Figure 1, V2 is the 8um of the volume oP the mixing chamber and the volume of the total vent space including the surge chamber. V
is V2 plus the volume of the measuring chamber. Other con~igurations will re~ult in compre~sions occurring in different parts of the apparatus, as shown in Figure 6 below for a different emhodiment.
In some e~bodiments o~ the invention, the formulas de~cribed above will not ~trictly apply. For example, even in the embodiment ~hown in Figure 1, a different mode of operation ca~ allow proper functioning of a ven~-assisted stop-flow junction without the indicated formulas being strictly adhaared to. For example, in Formula 1, the momentum of diluent flowing from diluent application site 170 can be used to overcome back pressure at the stop-flow junction even if the height of diluent and sample together are insufficient to start flow from an equilibrium state.
Alternatively, the various techniques described in U.S.
Patent No. 4,868,129 can be used to start flow rather than relying on the increased height of the column of sample and diluent. Other factor~ (such as controlling capillary action by varying the surface attraction of housing wall~ to liquid sample) can also be used in designing properly ~ized and shaped channels. However, use of these formulas in producing an initial design, 030191 19.

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followed by emperical optimization i~ preferred over attempts to calculate a design from physical principles.
Essential characteristics of this aspect of the invention, however, are that compression o~ trapped gas that would otherwise escape through a vent takes place in an enclosed space created by closing all vents to that space and that the advancing liquid supplys the compressive force), with the resulting increase in internal gas pressure being used to oppose the flow o~
sample past the stop-flow junction under consideration.
The increase in internal pressure is not itself sufficient to stop flow in the absence of a stop-flow ~unction at the location where stoppage of flow i~
desired. It should be noted that an internal gas pressure equal or even slightly greater than the head pressure, while sufficient to maintain an equilibrium state once flow is stopped, is not necessarily sufficient by itself to stop flow of a moving liquid (because of forward-ba~ed capillary action and momentum3 in the absence of the back pressure created at the stop-flow junction.

C. Stop-flow ~unction nozzle An additional feature that can be used to increase stability of the stop-flow ~unction is to provide a nozzle surrounding the capillary por~ion of the stop-flow junction that pro~ects into the non-capillary region (which can include projection into a recessed area in a chamber wall). The nozzle is shaped so as to provide exterior nozzle urfaces which form an acute angle with the adjacent surfaces of the interior wall of the capillary passageway. A typical pro~ection with acute angles to prevent creep of liquids around the edges of the stop-flow junction and to increase the practical amount of available back pressure is shown in Figure 2.

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Figure 2 is an expanded cros~3-section of the 19 8 area surrounding stop-flow ~unction 145 ~hown in Figure 1. Measuring chamber 140 is visible a:Long with the stop-flow junction 145 at the point where capillary s chamber 140 enters non-capillary mixing chamber 150.
Housing walls 141 surrounding the opening at 145 pro~ect into chamber 150. Surface 142 of the wall forms an acute angle (represented by ~) with the ad~acent interior wall of measuring chamber 140. The preferred shape for the nozzle formed by walls 141 is a cone when stop-flow junction 145 i5 circular. Howe~er, there arè
no particular limitations on the shape of the nozzle as long as an acute angle is maintained. A cone recessed into a surface of a non-capillary chamber is preferred, as shown in Figures 7B-7D below, when liquid flow or other motion (such as of a mixing element) occurs in a chamber containing a stop-flow ~unction.

D. Through-body stoP-1Ow ~unction Still further improvements in stability of the stop-flow junction can be achieved by forming the stop-flow junction from a single housing body member rather than forming it at the junction of two members used to form a ca~ity. Such stop-flow junctions are referred to as through-body stop-flow ~unctions.
Earlier patents have de~cribed the formation of apparatuses containing stop-flow junctions. These earlier patent~ and related applications ha~e described stop-flow junctions as occurring at the ~unction between two body members that formed the internal cavities of the apparatus in which the ~unction is located. For example, Figures 3A and 3B show prior-art stop-flow junctions formed a~ an intersection between (1) a body membsr in which the various capillary and non-capillary chambers are formed as depressions on ~ surface and (2) a second body member that encloses the depressions in the surface of the first body member to form the 030191 21.

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interior chambers. In Figure 3A, capillary channel 140 and chamber 150 ara visible in body member 102, while body member 104, when sealed to body member 102, turns the depressions originally on the surface of body member 106 into internal chambers.
However, the inherent: problems of sealing one body member completely to another can sometimes cause unanticipated failures of the stop-flow ~unction. As shown in Figure 3B, which is a cro s-sectional view taken along lines B-B of Figure 3A, joint 154 between body members 102 and 104 intersects with the opening of capillary chamber 140 in wall 152 of chamber 150. If ~oint 154 is completely sealed, no problems arisa.
However, if during the manufacturing process, a gap is le~t at ~oint 154, capillary action will draw liquid in capillary chamber 140 into the crack and tend to defeat the purpose of stop-flow ~unction 145. Capillary ~creep~' will cause flow to occur at the edges of stop-flow ~unction 145, thereby allowing liquid to enter chamber 150.
This potential problem can be avoided by using a through-body stop-flow junction as shown in Yigure 4.
In this embodiment of the invention, measuring chamber 140 enters mixing chamber 150 not at a ~unction between two body members, but entirely within a single body member. As shown in Figure 4, a diluting apparatus is made up of three body members, namely a central body member 102 that contain~ various depressions such as 140a and 150 that will form capillary channels and non-capillary chambers when enclosed by additional body members 104 and 106. In this case, measuring chamber 140 comprises ~wo segments 140a and 140b. Segment 140b is formed in an injection molding process using a pin that passes through the mold used to prepare body member 102. Thus, when body members 104 and 106 are ssaled to body member 102 to form the final apparatus, no ~oint between two or more body members exists at stop-flow 030191 2~.

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junction 145, and a sharp edge is maintained around the entire perimeter of the stop-flow junction.

E. Rupture ~unctions as stop-flow 1unction protectors Diluters that operate using stop-flow junctions of the invention can be prepared using multiple stop-flow junctions in which onP of the junctions is sacrificial; i.e., it is designed to fail before other junctions in order to protect the operation of the other junctions. Such a sacrificial ~unction i~
referred to as a rupture junction in this speci~ication.
For example, a number of preferred embodiments in which stop-flow ~unction~ of khe invention can be used contain valves that are operated by the application of an external force to the valve (~ee U.S.
Application Serial No. 117,791, filed November 5, 1987).
However, the opening and closing of valves in a diluter causes pressure waves to travel through the fluid contained in various passageways in the device.
These pressure waves can cause the failure of a stop-flow junction. By providing a rupture junction designed to fail at a pressure lower than the maximum back pressure that is available at other stop-flow ~unctions in the same capillary passageway, relief for the pressure wave is provided in a manner that will not adversely affect the operation of the diluter or other apparatus. For example, a capillary passageway can be provided containing a valve in some portion of the passageway. If it is desirable to retain liquid in the - capillary passageway on one side of the valve location, a rupture junction (i.e., a stop-flow iunction with a lower maximum back pressure) can be pro~ided on the other side of the valve in the same capillary passageway. Thust when the valve is closed, any pressure wave will be relieved by the failure of the rupture junction prior to failure of the stop flow 030191 23.

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junction that is designed to hold. Preferably the maximum back pressure available at the rupture junction will be at least 10% less than the back pressure available at the next weakest stop-flow junction in the passageway, more preferably at least 20% less.
Such an example is shown in Figure 5. By now, many of the common features of this diluter will be recognized. Diluter 100 contaiLns an application site 110, a capillary passageway 120 leading ~rom sample application site 110 to measuriLng chamber 140 comprising segments 140a and 140b. Segment 140a terminates at stop-flow junction 146 where the segment meets diluent application site 170 while segment 140b terminates at stop-flow junction 145 at the entrance to chamber 150.
Vent 101 and vent closing means 105 are present as in Figure 1. A valve is present in the capillary passageway leading to measuring chamber 140, which consists of flexible wall member 201 and plunger 202, which i~ exkernal to the device 100 and which operates to force flexible wall member 201 against the opposing wal} to block passage of fluid. A rupture junction is present at 147 in capillary pas~age 120 leading to measuring chamber 140. Rup~ure junc~ion 147 leads into rupture chamber 148 (for containing excess liquid) which is vented by vent channel 149.
In operation, sample applied to sample application site llO flows through capillary channel 120 and fills all of the capillary spaces between the application site itself and stop-flow junction~ 145, 146, and 147 (the last being the rupture junction).
When plunger 202 is activated to close the valve, a pressure wave is generated in the capillary passageway.
Since stop-flow junction (rupture ~unction~ 147 is designed to fail before either stop-flow ~unction 145 or stop-flow junction 146, the pressure wave generated by closing the valve is relieved b~ flow of excess sample into rupture tank 148.

030191 24.

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Although the rupture junction has been described with regard to a particular embodiment shown in Figure 5, other embodiments will be readily apparent to those skilled in the art. Rupture junctions can be designed into any apparatus in which temporary halt of flow is desired after which events occur that are not intended to, but which may acc.identally, cause flow to occur at stop-flow ~unctions, such as the opening and closing of vaxious valves. By providing a location at which pressure can be relieved without adversely affecting locations where flow should still be arrested, rupture junctions provide additional stability to device~ containing stop-flow junctions of the invention.

II. Integration of stop-flow ~unctions into a diluter A. Components of diluter other than the stop-flow junctions As with the apparatuse~ described in U.S.
Application Serial No~. 090,026 and 117,791, the cartridges of the present invention include a sample application site, a diluent application site, a measuring chamber, a mixing ~receiving) chamber, various channels to provide flow of liquid between parts, and, in the case of serial diluter~, a mixture isolating and measuring chamber and at least one valve controlling passage of fluid from the mixing chamber to the mixture isolating and measuxing chamber. All of these parts of the cartridge have been described in the indicated applications~ which can be referred to for greater detail.
The apparatus of the invention can provide for a single dilution, as in the valveless diluter described in U.S. application serial No. 090,026.
Serial dilutions can be provided for using a valve to control passage of a portion of the initially obtained mixture into a mixture isolating and measuring chamber.

~0299751 030191 25.

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This mixture isolating chamber can take any of the forms described in U.S. Application Serial No. 117,791.
However, in preferred embodiments as described herein, the mixture isolating chamber contains essentially the same chambers and passageways as the initial diluting pathway described above. All of these parts are described in greater detail be:Low. The following detailed description of the vaxious parts of the apparatus is organized by following the course of action as a sample is applied to the apparatus and is diluted.

(1) Sample The sample i5 a liquid and may be derived from any source, such as a physiological fluid; e.g., whole blood, blood plasma, blood serum, saliva, ocular lens fluid, cerebral spinal fluid, pus, sweat, exudate, urine, milk, or the like. The liquid sample may be subjected to prior treatment, such as preparing serum or plasma from blood or dissolving or suspending a solid in a liquid. Examples of sample treatments prior to application to the apparatus o~ the invention include concentration, filtration, distillation, dialysis, inactivation of natural components~ chromatography, and addition of reagents. In addition to physiological fluid~, other liquid ~ample~ can be employed. Examples of other liquid samples include process streams, water, plant fluids, chemical reaction media, biological growth media, and the like. For the most part, the liquid will be aqueous, although other liquids can be employed.
Aqueous media may contain additional miscible liquids, particularly oxygenated organic sol~ents, such as lower alkanols, dimethyl formamide, dimethyl sulfoxide, acetone, and the like. Usually the solvents will be present in less than about 40 vol~, more usually in less than about 20 vol%, in order to maintain the high surface tension that is present in aqueous solutions.
However, the apparatus of the invention can be modified 030191 26.

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2~198~
as described below for use with liquids exhibiting different surface tensions.
The apparatus a~ described initially herein provides for a single dilution of a sample with a diluent. Any apparatus that carrie~ out a dilution in the manner described i8 considered to ~all within ~he scope of the present invention, whether the dilution occurs by itself or as part of additional operations that occur in the device. For example, other operations can be carried out on an oriyinal sample so as to provide a mixture. This mixture is then the "sample"
that i5 later diluted. Alternatively, provision can be made for other operations to take place on the mixture formed in the manner described above.
(2) Sample application site The sample application site (also referred to as a sample receiving site) will ~enerally be a cavity on a surface of the apparatus or may simply be an opening (optionally surrounded by a ring or tube) lead-ing to the i~terior of the apparatus. The æample application site can contain a filter, for example, to separate red blood cells from plasma (see U.S. Patent No. 4,753,776), or may represent a connection between the apparatus of ~he invention and some other apparatus that manipulates ~he sample prior to its entering the present dilution apparatus. For example, the application site can be a reces~ into which a standard capillary tube will fit.
When the sample application site is a recess for insertion of a capillary tube, the capillary tube can act either as a convenient means for transferring the sample or can act as a measuring chamber, either by completely filling the capillary or by filling the cap-illary to a particular mark. The sample application site in such lembodiments acts as a point of transfer.

030191 27.

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In other ca~e~, the ~ample application ~ite will be an external chc~mber, such a8 a rece8s on an upper surface of the de~ice into which ~clmple is in-serted. Such sur~ace reces~es are raferred to horein a~
external chambers, to di~tinguish them from chc~mber~
located in the interior of the hou~ing that form~ the cartridge. The spp~ication site can be provided wlth a rai~ed lip surrounded by a catch ba~i~ so that tha application site can be filled to overflowing with exce~s sc~mplQ overflowin~ into the catch ba~in. Mean~
for draining off a large exces~ of aample or ~ample inadvertently applied to the wrong location are discus~ed in U.S. applic~tion ~erial numbers 090,026 and 117,791, discussed abova.
(3) Capillary pas~aqewaYs, includin~
mea~urement chc~mber _ When sc~mple i3 applied to the sample applica-tion site, the li~uid sc~mple normally flow~ without the application of external ~orce (except unasslsted grav-ity~ through a fluid pa~sageway into a measurinq chamber in the interior of the devlce. ~ de~cribed in U.S.
Patant No. 4,868,129 and U.S. Application Serial No.
117,791, the sc~mple can flow directly into a measuring chambar. ~OWOV4X, it i~ alBO possible for ths ~ample to flow into a flow directing chc~mber, comprising an internal chc~mber in the housing that form~ the apparatus before enterlng a measuring chambar, as described in U.~. Application 5erial No. 337,286, filed April 13, 1989. External force, e.g., from compre~sed air, can be u~ed to mo~e the 8ample to the mQasuring or flow directing chamber but is not required and in fact i~ not preferred. The flow directing chamber (when pre~ent) act~ to divert a portion of the ~ample that fir~t entere the flow directing chamber into the sample : mea~uring chc~nber, which has a predetermined volume and which operates to measure and hold a portion of the 0301gl 2~.

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sample for dilution. ~he remainder of the sample th ~ 619 enters the ~low directing chamber is automatically diverted by the flow directing chamber into an exit port leading to a waste chamber or to some other means of disposing of excess sample beyond that required to fill the sample measuring chamber.
Flow directing chambers and the various appurtenances thereto, such as waste exits and waste chambers, are described in detail in U.S. Application Serial No. 337,286 (above). However, since flow dirscting chambers are not used in preferred embodiments of the device containing improved flow-stop ~unctions of the present invention, the reader is referred to the earlier application for a complete description of this type of fluid passageway.
The measuring chamber can be a capillary channel or chamber, in which case capillary action will aid or in some case~ provide all the force necessary for filliny the measuring chamber with sample from the sample application site by way of the flow directing chamber. Capillary channels and chambers will generally have at least one dimension perpendicular to the flowpath in the range 0.01 to 2.0 mm, more generally 0.1 to 1.0 mm. Capillary spaces (of whatever t~pe) have at 2S least one dimension at right angles to the direction of flow in the range required to ~uppor~ capillary flow.
Capillary channels have both dimensions at right angles to the direction of flow in the range required to suppor~ flow. Capillary chambers have one dimension at right angles to flow that would not support capillary flow but provide for capillary flow by having the second dimension at right angles to flow in the required range (similar to the space between two flat plates that are closely spaced). However, larger measuring chambers that are no~ capillary in any dimension are also possible. The sample measuring site is said to be in "fluid receiving relationship" to the previous capillary passageways in order to indicate that unassisted flow 030191 ~9.

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206198~
into the measuring chamber occurs. In order for proper operation of the stop-flow ~unction to occur, it is essential that the measuring chamber be filed solely by capillary and gravitational forces, as will be apparent from the description of the ~top-flow junction below.
It should be noted that internal spaces of a diluter that can be of either capillary or non-capillary dimensions, such as the measuring chamber, are referred to herein as "chambers" without regard to whether they are capillary channels, capillary chambers, or non-capillary chambers, in order to avoid awkward repetitive language. When the specific dimensions are important, specific lanyuage, such as "capillary chamber," is used in place of the more general "chamber." In other cases limitations on the type of space ~capillary or non-capillary) that is under con3ideration will be apparent from the context and from the functional requirements of the space.
The geometry of the measuring chamber is such that, when diluent is added to the apparatus at a later dilution step after measurement is completed, essentially all of the sample in the measuring chamber will be expelled into the mixing chamber. One means of accomplishing this is by providing for smooth flow of diluent through the measuring chamber. A straight or curved tube with an e~sentially constant cros~ section open at both ends is thus a preferred embodiment for this type of measuring chamber. Thi~ type of measuring chamber is seen in measuring chamber 140 of Figure 1.
In preferred embodiments of this type, diluent enters the measuring chamber in a front across the entire cross-sectional area of flow. This helps avoid mixing of diluent with sample and passage of diluent through the measuring chamber without expelling essentially all of the sample, which can occur if a small stream of diluent enters into a broader cross-sectional area of the measuring chamber.

030191 30.

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However, measuring chambers that vary in cross section are also possible, as discussed in prior applica-tions. Nevertheless, it is desirable to have the initial portion of the measuring chamber be as ~mall as practical, as this aids in reducing the amount of sample that may be lost from the measuring chamber when diluent initially rushes into the flow directing chamber.
Initial diameters of less than 0.5 mm are desirable, preferably less than 0.2 mm. If the entrance to the sample measuring chamber is large, sample can be washed up into other passageways or chambers when diluent first enters. An unmeasured quantity of sample then flows, e.g., into a waste chamber as diluent continues to fill a flow directing chamber and then flow into both the measuring chamber and the waste chamher. Although this problam cannot be completely eliminated, using a small opening to the sample measuring chamber will reduce sample losses to acceptable levels. A small opening is therefore preferred even when the remainder of the measuring chamber is large ~e.g., of non-capillary dimensions).
Additionally, while most measuring chamber~
will be manufactured to have a fixed volume, it is possible to provide chambers (both measuring chambers and other types of chambers and internal compartments) whose volume can be varied, for example by a closely fitting plunger used to ad~ust the volume of the chamber prior ~o use. The internal volume of such an adjustable chamber would be set to the desired value by the user, normally prior to addition of sample to the apparatus.
When sample flows into a measuring chamber, flow stops when sample reaches a stop-flow junction, as has been described in earlier applications.

(4) Diluent apPlication site A number of diluent application (diluent receiving) sites are disclosed in U.S. Patent ~o.

030191 31.

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4,868,129 and U.S. Application Serial No. 117,791, discussed above. Any of these diluent application sites can be used in an apparatus of the present invention if desired. In the most preferxed embodiment, the diluent application site i~ an internal vented chamber in the housing that forms the apparatus. Located in the chamber is a rupturable container of diluent. Glass containers are particularly pre~erred, although frangible plastic can also be used. An access port may be provided so tha~ externally applied pressure can be used to rupture the container. However, it i5 not necessary to provide an access port, since a frangible glass or plastic container located within the housing can be hroken by a sharp blow to the housing itself. If the frangible container is -qized for its charnber 50 that deformation o~ the chamber walls (i.e., wall of the housing surrounding the frangihle diluent container) allows the motive force of the blow to also strike the frangible container, then the frangible container will break without requiring an access hole to the chamber.
This represents an improvement over prior embodiments of the diluter, as leakage of diluent from the cartridge after use is eliminated. If desiredr a flexible area can be provided on the wall of the chamber surrounding the diluent container, such as by providing a thin housing in a target region at that location. Providing a thinner and more flexible honsing will increase the possible deformation upon receipt of a blow. The central point of ~he target region can be thicker than the surrounding flexible region in order to better absorb the ener~y of the blow without breaking.
Exact dimensions are best determined emperically for a given diluent container, chamber, and housing material. As an example, an ABS housing with a wall thickness of 0.020 inch, a target region thickness of 0.015 inch, and an ampule chamber 0.275 inch thick containing s:lass ampules ranging in thickness from 0.258 to 0.272 inch, worked well.

030191 32.

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A passageway connects the diluent chamber to the flow directing chamber or measuring chamber.
Diluen-t flows into the measuring chamber so that the hydrostatic pressure at the stop-flow junction i~
S exceeded and the sample is expelled into the receiving chamber along with a portion of the diluent. Excess diluent flows into a waste chamber in some embodiments or remains in the diluent application chamber and/or flow directing chamber.

(5) Mixinq chamber There are no particular restraints on the geo-metry of the receiving (mixing) chamber other than that smooth fluid flow be provided for in order to prevent trapping of gas bubbles. Providing entry of sample and diluent into a lower portion of the receiving chamber and providing an upper surface of the receiving chamber that slopes upward toward a vent both aid in avoiding trapped bubbles. It is desirable, however, to ensure that the exit for mixed diluent and sample (if present in the receiving chamber; see below) is located at a distance from the entrance for sample and diluent. If the exit and entrance are located too close to each other, diluent flowing into the chamber while mixture is exitin~ can reach the exit too early and result in diluent rather than mixture reaching the second measuring chamber. Other provisions can be made to ensure smooth flow of mixture through the exit, such as locating the mixture exit at a low location and the diluent entrance at a high location for diluents that are less dense than the mixture of sample and diluent (and vice vexsa).

0301gl 33.

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(6) Vents The vents used in the various chambers of the device can merely be a small hole terminated by a stop-S flow junction in order to avoid exit of liquid from thedevice or can be a more sophisticated ~ent de~igned for gas exit without exit of liquid (e.g., a microporous, hydrophobic plug capable of passing air but not hydrophilic liquids~. Stop-flow junctions can also be placed in the early portion o~ a long vent to preven~ a relatively large quantity of liquid from entering the vent from the vented chamber. A vent or other means to allow exit of trapped air i5 provided at every location in the apparatus in which the trapping of air would interfere with the passage of liquids between the various chambers and/or channels of the device. If desired vents can be salectively opened and clo~ed, as described for vent-assisted stop-flow ~unctions.
A preferred manner of forming vents is to use interior waste space in the housing as vent space to catch any liquids that may accidently be forced through a vent channel. ~he initial venting channel leading from, for example, a mixing chamber to the waste space is then essentially an internal venting space, with an external vent at a location in the waste space that is unlikely to be reached by liquid which can function as the final external vent. In preferred embodiments/ this internal~external venting system can al~o pro~ide the surge tank arrangement already discussed for vent-assisted stop-flow ~unctions, in addition to pro~iding the additional safety function of trappping potentially dangerous samples or reagen 9 inside the housing (which can be disposable).

(7) Size of chambers and capillaries Although there is no theoretical upper limit on the size of samples that can be measured and diluted 030191 34.

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in this fir.st step ~or later steps) using an apparatus of the invention, the method and apparatus ~re particu-larly suitable for measuring and diluting small quanti-ties of liquids. Accordingly, the sample measuring chamber will generally have a volume of from O.l ~L to 100 ~L, preferably 1 ~L ~o 30 ~, and most preferably 3 ~L to 10 ~L. The receiving chamber, which acts to limit diluent volume and fix the ratio of sample to diluent, generally has a volume of from 3 ~L to 1000 ~L, preferably 10 ~L to 300 ~L, ancl most preferably 30 ~L to 200 ~L, thereby pro~iding dilution ratios o~ from 104:1 to 3:1, preferably 103:1 to 4:1, and mo~t pre~er~bly 100 :1 to 5 :1. Channels through which capillary flow will take place will u~ually have opposing walls spaced in the range of about 0.01 mm to 2 mm, more u~ually about O.l mm to 1 mm. The capillary space3 can be tubular (which does not necessarily imply a circular crosssection but can be square or other regular shapes) or can represent the space formed by flat plates and side walls with the side wall~ being spaced further apart than a capillary distance. A tubular chamber with at least one flat side te.g., a square cross-sectional area/ a rectangle with ad~acent sides di~fering in length by no more than a factor of 1:2 to 1:4, or a semicircular chamber) are preferred for ease of manu-facture in cases wh~re channels are being formed by the joining of two ad~acent surfaces, one of which can be flat.
It should be recogniz~d that statements in this specification indicating upper and lower limits of range~ are to be taken as individually designating a series of upper limits and a series of lower limits which can be utilized in any combination. For example, a typical upper limit and a preferred lower limit may be used in combination to define a range of intermPdiate preference.

030191 35.

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2 0 ~ 4 (8) Valves Any type of valve that will control the pa~-S sage of liquids between chambexs and/or channels can be used in ~he apparatus of the present invention. Simple valves that can be actuated to move between an open and a closed position by the application and release of a simple external force are preferredO
Examples of such valves include resilient blocking members that are pres~3nt in or ad~acent to a liquid flowpath. For example, a re~ilient blocking member can be present in a converging or diverging pathway so that the narrow portion of the pathway i~
blocked by the resilient blocking member when the blocking member is in its normal po~ition. Application of force in a direction generally away ~ro~ the re-stricted portion of the flowpath and toward the wider portion of the flowpath will open the valve by moving the blocking member away from the narrow walls of the flowpath. Alternatively, a normally open valve can be provided which is blocked by movement of a resilient blocking member to a location that cut~ off flow of liquid. Specific examples of such valves are set forth in more detail below.
Other examples of such valvss include sliding pins closely engaging a channel that laterally traverses a fluid flowpath. The pin has a segment capable of obstructing flow through the flowpath when the pin is in a first position and a ~egment capable of allowing flow through the flowpath when the pin is in a second position. Examples of such pins include rectangular pins having a flowpath channel betwe~n two opposite faces of the pin, the flowpath channel being out of register when the block is in a closed po~ition and in rPgister with the prlncipal flowpath when the block valve is open. Pins with circular cross-sections can be used by prov;ding an obstructing segment of the pin that 030191 3~.

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2~fi~8~ -snugly engages the channel in which the pin fits and obstructs the flowpath when the pin is i.n a closed position. A smaller cross-sectional area (such as is present in the handle of a dumbbell) provides an annular flowpath around the smaller, central portion of the pin when the pin valve is in the open position.
A resilient member can be provided to bias the pin into either the closed or t:he open position. A
force acting on the pin can then slide the pin to a second location S4 that the pin valve is in the alter-nate position.
In preferred embodiments, access for the ap-plication of an external force on the pin is provided so that the pin can be moved between its two positions.
For example, a section of the pin that protrudes exter-nally from the apparatus can be provided so that a force acting parallel to the sliding axis of the pin can move the pin from its first biased position to a ~econd po-sition by acting against the direction of the biasing force. Alternatively, an aperture leading from a face of the pin opposite the biasing force to the external envi.ronment can be provided. Externally applied pres sure, such as from compressed air or a finger of an ex-ternal apparatus ~hat enters the aperture, can be used to slide the pin between its open and closed positions.
A resilient seal can be provided to prev nt loss of liquid through the aperture while allowing force to be applied to the pin. Such seals can also be provided for the resilient blocking member~ described above.
The valves that can be used as integral parts of a cartridge of the present invention are not limited to those specifically exemplified here. Rather, any valve can be used that can control the flow of liquids through small flowpaths, such as flexible walls (e.g., latex) of a flowpath that can be compressed to restrict flow of liqui.d through the flowpath. Additionally, a dissolvable barrier can be provided in instances where 030191 37.

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It is also possible to provide an external valve. For example, a flowpath through which capillary flow occurs can be blocked by closing an external vent.
When the external vent is closed, liquid cannot enter ~he capillary pathway because of aix or other gases in the capillary pathway. Opening the vent allows liquid to enter the capillary pathway. If the vent is closed while liquid i5 contained in the capillary pathway, the isolated liquid can later be used for other manipulations.
Valves con~isting of external vent controls can be used in any situation where flow occurs through a capillary pathway (so that trapped air is e~fective to control flow of liquids) and wh~re no free liquid that might leak is stored in the cartridge prior to u~e.
Encapsulated liquid (e.g., in glas~ ampules) can be present in devices using external vent control~. In many cases it is desirable to store pre-measured diluents (which can contain reagents) in the cartridge when the cartridge is delivered to an end user.
Internal mechanical valves or rupturable barriers axe preferred for such uses in order to prevent accidental leakage.
By providing valves that can be operated by a simple externally applied force, a cartridge like device can be provided in which the valves are opened and closed in a predetermined manner by an analytical device into which the cartridge is inserted~ This analytical device can contain various optical and/or other types of sensors for detecting the presence of liquids or analyte~ in various mixing and/or measuring chambers of the cartridge in addition to providing means for opening and closing the valves and is therefore sometimes referred to :in this specification as a monitor.

030191 38.

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-: - , 2 ~ 8 4 (9) Reagents and assays The apparatus of the present invention can be designed for us~ with a particular assay or can be designed and prepared as an apparatus in which multiple assays can be carried out, depending on the order in which various valves are opened and closed and the contents of the various diluents, which can contain reagents for the development of a detectable signal (e.g., a color reaction) that depends on the presence of an analyte in the sample.
Reagents can be provided at various locations in th~ device. Incubation time~ can be controlled by either manual operation of valves or by a mechanically or electronically stored program in the monitor into which the cartrid~e is inserted. The program would control the order and timing of opening and closing valve~. The programmed device would contain solenoid~
or other mean~ for providing force to open and/or close valves or rupture contain~rs containing diluent. In embodiments in which flow through a capillary pathway is being controlled by the openiny and closing of a vent, a movable sealing pad that is capable of closing the vent will form part of the external programmed device into which the cartridge i9 inserted.

(10) Monitor The appaxatus shown in the Figures and otherwise described herein will normally be inserted into an apparatus in which analytial measurement~ on the sample can made. The analytical instrument is sometimes referred to as a monitor. Optical measurements are common and are the preferred type of mea~uremQnt for use in monitors. A light source and a detector are located in the monitor so that the light impinges on the desired location in the mixing and dilution chamber, passes 030191 39.

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through the chamber and the material enclosed therein, and impinges on the detector :Located at -the other side of the cartridge. This i5 accomplished by inserting the cartridge into a chamber of the monito:r so that all of the parts are placed into proper registration with each other. The present invention requires nothing new in the way of light sources, detectors, and registration means, since all spectrophotometers that engage cu~ettes and carry out light measurements there through provide the necessary detection and registrakion sy~tems.
However, the monitor can proved additional light sources and detectors to detect the presence of the fluid at various points in the fluid pathways throughout the cartridge. In this specification such components are called system control components since they represent a means by which the monitor can ~erify whether sample, the diluted mixture, or the like have reached the proper points in the fluid pathway in the proper sequence and at the proper time. For example, light sources and detectors can be placed at opposite sides of the cartridge so that the detector measures light passing through the sample in passageway 120 at optical window 122 to determine when s~mple has been applied to the cartridge (see Figure 6). Various operations of the cartridge can be automatically provided by detecting presence of absence of various liquids in the cartridge, as has been described in pre~iously listed applications and patents.
The monitor is generally designed to be capable of detecting correct operation of the cartridge by providing sensors that detect the presence of liquids at numerous locations in the fluid pathways of the cartridge and comparing the signals provided by the sensors with the signals that would be produced during proper operation of ~he cartridge. Automatic detection of proper operation is desirable when ~he cartxidge i5 in the hands of an unskilled user, which is a de~ired end use of the cartridge. For example, if the user must 030191 40.

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apply a drop of blood (~9 the sample) to the sample application site, several problems can occur. Some patients have trouble obtaining a drop of blood of sufficient volume. For example, if proper operation of the cartridge requires 25 ~1 of blood and only 20 ~L is added to the sample application site, the sample measuring site may not completely fill. If diluent i5 then added automatically (such as after a preselected time), the dilution will be greater than desired, and an incorrect result will be obtained.

(11) Construction The car~ridges of the invention are typically prepared from molded plastic a~ described in U.S. Patent No. 4,756,844, the only principal differences between the production methods described in the patent and the production required for the present apparatus being in the mold used to form the ~arious chambers. Aæ
indicated in the patent, plasma ekching can be used to improve flow characteristics through the various capillary pathways, since most molding plastics are hydrophobic and need to be rendered hydrophilic for reproducible capillary flow to occur.

(12) Second sta~e of diluter In particular, the present inventors contemplate providing serial dilution and mixing capabilities using a mixture measuring and isolating chamber hydrostatically connected to the mixing chamber and a valve controlling passage of fluids from the mixing chamber to the mixture isolating chamber. The first dilution takes place as indicated above during which time the valve is closed to prevent escape of liquid from the mixing chamber. After the first mixture is formed, the valve controlling flow to the mixture 030191 41.

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i~olating and measuring chamber is opened, and fluid flows from the mixing chamber under the influerlce o~
hydrostatic pressure and/or capillary attraction. The portion of the mixture isolating chamber into which the mixture flows is smaller in volume than the total volume of mixed sample and diluent. This volume is determined by the geometry of the chamber, the amount of hydrostatic pressure available from liquid in the mixing chamber, and any capillary forces that are present.
U.S. Patent Application Serial No. 117,791, described above, describes various geometries that can be provided for a mixture isolating chamber depending on whether the intent is to carry out a second dilution in the orlginal mixing chamber o to transport the i~olated portion of the mixed sample and diluent to another location for further dilution and~or analysis. Any apparatus that carries out a ~ingle dilution as de~cribed above and a second dilution as described in the prior application will fall within the scope of the present invention.
However, a particularly pre~erred embodiment of the present invention i~ directed to an apparatus in which serial dilutions are carried out, bo~h of which fall within the scope of the single-dilution invention set forth above. In such embodiments, the mixture isolating chamber will comprise the same types of chambers and passageways as described previou31y, with the exception that they will operate on the mixture as a sample rather than on an initially obtained sample.

B. SPeci~ic example of ap~atus (1). Descri~tion of exemplary appaxatus A series of Figures is provided to illus~rate a particularly preferred embodiment of the invention.
The embodiments shown in the Figures are not intended to be comprehensive, and numerous other embodiments within 202~9751 030191 42.

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the scope of the appended clai.ms w:ill be apparent to those of ordinary skill in the fiald of the in~ention.
Figure 6 is a plan view from th~ front of a first embodiment of the inventlon in which lines A-A, D-D, etc., show the location oE the corresponding cross-sectional views shown in Figures 7A, 7D, etc. ~s shown in Figure 7A~ housing 100 is p.repared from three separate pieces, a central base member 102 and two cover plates 104 and 106. Chambers formed in the front face of base member 102 are shown w.ith solid lines in Figure 6. Passageways formed in the back face o:E base member 102 are shown by dashed line~ in Figure 6. Through connections, which are generally holes pa~sing from one face to the other, are shown by circles in Figure 6.
All such passageways would be visible in embodiments prepared from transparent plastic, as de~cribed in U.S.
Patent No. 4,756,844. However it i~ also posslble to prepare the cartridge from an opaque material if provisions are made for light paths at the appropriate locations.
The apparatus shown in Figure 6 is capable of carrying out two d.ilutions serially. Parts of the apparatus associated with the first dilution are numbered from 110 to 182. Parts of the apparatus associated with the ~econd dilution are numbered from 205 to 282. Where two parts perform the same function in the first and second dilutions, the las~ two digits of the identifying number are the s~me. Parts of the apparatus associated with the housing are numbered from 100-106. The apparatus will be described hy reference to the indicated numbers while following a sample through a series of two dilutions in the apparatus.
A sample is added initially to sample application site 110. The sample flows down capilla~y passageway 120 to measuring chamber 14Q. Passa~eway 120 consists of an inital ~egment 120a connecting diluent application site 110 to the remainder of the passageway, a se~ment 120b (leading to a rupture junction 147 shown 030191 43.

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in Figure 7I), and a qegment 120c containiny valve 125 that is connected at one end to bo-th segment 120a and 120b and at the other end to measuring chamber 140.
Segment 120b terminates in rupture chamber 130, which has a venting exit 132 and venting channel 133 leading to internal vent 134.
Sample continues to flow into and fill measuring chamber 140, which is of capillary dimensions. Measuring chamber 140 consists of ~ertical segment 140a terminating at stop-~low junction 146 and horizontal segments 140b and 140r (the latter terminating at stop-flow ~unction 145 as shown in Figure 7B). Sample flow stops when the leading edge of the sample reaches the various stop-flow junctions 145-147.
Vent channel 152, located in a upper portion of dilution and mixing chamber 150, is connected to vent surge tank 154 and eventually to vent opening 156 by channel 153 to allow controlled exit of gases from chamber 150.
Frangible container 175 is (not visible in this view) provided in an internal chamber 170 that functions as the diluent application site. Chamber 170 is connected by internal passageway 180 to measuring chamber 140 at stop-flow junction 146. Passageway 180 is vented to atmosphere via a vent channel 182 leading to an in~ernal vent 182.
Mixing in chamber 150 can be provided by a number of techniques, such as are described in a co-pending application entitled ~Reciprocal Nixing Cartridge," filed April 6, 1989, assigned Serial No.
334,304. It is possible to begin mixing the sample and diluent as they enter the chamber 50 that any mixture entering the vent will have approximately the same composition as the mixture remaining in the chamber.
Better is to allow undisturbed filling of the chamber.
In either event, the volume of the vent is sufficiently small so that negligible Prror result~. Additionally, it is possib:Le to include a separats stop-flow junction in the vent channel to prevent excess exit of liquid, 030191 44.

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should higher accuracy be desired. Such a stop-flow junction in the vent channel exiting the mixing chamber is shown ~elow in Figure 7G.
Exit 210 in receiving chamber 150 serve~ as the entrance for mixture into t:he second dilution portion of the apparatus. During the first dilution, however, pas~ageway 225 is bloc:ked by valve 225, and trapped air prevents mixture from entering ~he passageway. When the valve is opsn, a portion of the mixture flows through exit 210 and channel 220 to a second measurement chamber 240/ referred to herein as the mixture measurement chamber, which, as for measurment chamber 140 r consists of a vertical segment 240a and horizontal segments 240b and 240c. Mixture measurement chamber 240 terminates at stop flow ~unctlon 245 where chamber 240 intersects with mixture diluting chamber 250 and at stop-flow ~unction 246 at the diluent end of the measurement chamber.
Second diluent i5 provided in rupturable diluent container 275 (not visible in this view) contained in diluent chamber 270. Diluent becomes available at diluent applica~ion site 270 upon rupture o the container, flows into channel 280, and enters mixture measurement chamber 240 at stop-flow junction 246. Channel 280 i8 vented at ven~ 282. As with the first dilution, the hydros~atic pressure provided by the diluent is available to overcome the back pressure at stop-flow junction 245. Diluent flows through mixture measuring chamber 240 into mixture receiver chamber 250, expelling trapped air through vent exit 252 and channel 253 leading to surge chamber 254. Surge chamber 254 is provided to give ~he volume necessary for proper operation, as described above. Mixing takes place in mixiny chamber 250 in the same manner as in mixing chamber 150.
Figures 7A through 7J show a serie~ of cross-sectional views at different locations of the embodiment shown in Figure 6. As mentioned previously, the 030191 45.

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apparatus is as~embled by attaching cover plate~ 104 and 106 to central body member 102 in which the various chambers and passageways are formed. In Figures 7A-7H, the top sides of the ~igure represents the back face of the embodiment shown in Figure 6 and the bottom xide represents the front ~ace except when indicated otherwise.
Figure 7A is a sectional view of the embodiment shown in Figure 6 taken along line A-A, with the back of the embodiment of Figure 6 appearing at the top of Figure 7A. The three body members that make up housing 100 are visible in thi~3 figure. A central body member 102 has various depressions in its upper and lower surfaces (as viewed) along with through passageways from one surface to the other. Front (104) and back (106) face plates are ~ealed to the central body member 102 to form the internal cavaties that make up the capillary and non-capillary chambers and pa sage~
of the diluter.
Turning to the internal cavities on the left side of the figure and moving toward the right, cavity 105 plays no part in the operation of the diluter but is an internal cavity that prevents the central body member 102 from being unduely thick, ~hereby reducing time spent during molding operations. Rupture tank 130 is next visible along with segment 120b of capillary passageway 120 and the rupture ~unction 147 at the intersection of pasRageway 120c and chamber 130.
Passageway segment 120b is visible along the front face of body member 102. Initial segment 120a of passageway 120 (not visible in this view; see Figure 6) join~ the remainder of the passageway at the common junction between segments 120b and 120c.
The use of passageways on both faces of central body member 102 and through passages between faces to prepare capillary passageways can he seen in section 120c, 120d, 120e, 120f, and 120g of capillary passageway 120, along with valve 125. Segment 120c is 202g9751 030191 46.

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formed by a depression in the front face of body member 102 that is covered by face plate 106. Sebment 120d is a through passage between segment 120c and the depression thak forms the loction of valve 125. Valve 125 operates by application of external pressure to flexible covering 127, which blocks passage of fluid when forced into depression 125. Depression 125 is connected to depression 120e i:n the hack face of central body member 102, and from there to through passageway 120f that connects to the last segment of passageway 120, a depression 120g in the front face of body member 102.
Segment 120 g is connected to horizontal measuring segment 140b at a location about midway between through passageway 120f and through passageway 140c, which terminates at stop-flow ~unction 145 in mixing chamber 150. Vertical measuring se~ment 140a (not visible in this view; see Figure 6) also is connected to segment 120g and segment 140b at their common junction.
Passageway 220, which leads from mixing chamber 150 to measuring chamber 240, consists of segments 220a-220h and val~e depression 225. ~hese passageway segment~ function in essentially the same manner as the various sagments of pa~sageway 120.
Measuring chamber 240a (not visible; see Figure 6) and 240b both ~oin with the far end of seyment 220h.
Measuring segment 240b leads to through passageway 240c, which terminates in stop flow ~unction 245 at the entrance to chamber 250.
Figures 7B through 7H are expanded sectional views of different s~op-flow junctions.of the diluter embodiment of Figures 6 and 7A. Figure 7B/ taken along line B-B of Figure 7B, is capillary ~top-flow ~unction 145 in chamber lS0. ~hrough passageway 140c terminates in nozzle 141 located in a recessed area 142 of wall 143 of chamber 150. Figure 7C, taken along line C-C of Figure 7A, shows capillary stop-flow junction 245 in 030191 47.

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chamber 250. Through pa~sageway 240c terminate~ in nozzle 241 located in rece~s 242 of wall 243 of chamber ~50.
A typical upper stop flow junction is shown in Figure 7D, taken along line D-D of Figure 6. Through passageway 140a terminates in nozzle 181 lo~ated in recess 182 of wall 183 of chamber 180. Figur~ 7E, taken along line E-E of Figure 7A, shows rupture junction 146 in rupture chamber 130. Through passageway 120b terminates in nozzle 131 located in wall 133 of chamber 130.
Use of diff0rent dia~eters ko provide different maximum back pressure3 can be seen from a comparison of Figures 7D, 7B, and 7E, which respectively show an upper capillary stop-flow ~unction 146 that is designed never to break, a lower capillary stop-flow junction 145 that is de~igned to hold initially and then break when diluent is applied, and a rupture junction 147 that is de~igned to break before either of the other two. The qcale drawings show a small diameter for upper stop-flow junction 146 (Figure 7D), an intermediate diameter for lower ~top-flow junction 145 (7B), and a large diameter for rupturs ~unction 146 (7E).
Stop-flow ~unctions of thP invention are also present at other locations. Figure 7F, taken along line F-F of Figure 6, shows vent 182 terminating in an interior was~e space 186. A nozzle 181 is present to increase back pressure. Figure 7G, taken along line G-G
of Figure 6, shows a stop-flow junction 152 in the initial through pas~ageway 153a of vent channel 153 at the exit of mixing chamber 150. A similar stop-f~ow junction 252 is present in vent channel 253 at the exit of mixing chamber 250 (not shown in de~ail; s~e Figure 6). These two stop-flow ~unction~ act to reduce the amount of liquid that exits the mixing chambers, thereby providing for more accurate dilution and mixing steps.
Additional stop-flow junctions can be provided at locations in vent channels more distant from the mixing 030191 48.

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(or other liquid-containing) chamber for additional leakage protection, such as at through passageway 256 of vent channel 253. Figure 7H, taken along line H-H of Figure 6, shows this through passageway in detail.
Through passageway 253b traverses body member 102 ~rom the initial portion 253a of vent channel 253 on the back face of body member 102 to the later portion 253c on the front face. A stop-flow nozzle 251 is visible at the location where passageway 253b enters channel 253c.
Several construction features of the diluter are seen in Figures 7I and 7J. Figure 7I is a sectional view of the sample application site 110 taken along line I-I of Figure 6 showing sample application cavity 110 and an intial section of capillary passageway 120 along with interior waste chamber 112. Figure 7J i8 a sectional view of the diluent application chamber 270 taken along line J-J of Figure 6 showing diluent appl.ication ~ite (chamber) 270, an inikial portion of diluent channel 280, and a portion of surge tank 25~.
Diluent container 275 is visible in chamber 270. A pin 103 in body member 102 that fits into a hole 107 of body member 106 in order to insure proper registration of body members 102 and 106 during manufacture is also visible. A flexible target region 277 with a a thicker central target point 278, which is struck by an external blow in order to break container 275, is also visible in this view.
The entire appara~us shown in Figures 6 and 7 would be approximately 5 cm high and less than 8 cm wide with body member 102 being about 0.7 cm in thickness. The cartridge can readily be prepared in other sizes to carry out other analytical measurements.

2029g751 030191 49.

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(2) Operation of exemplary serial diluter Outlined below is a typical cartridge operating sequence with reference to the embodiment shown in Figures 6 and 7:
(1) A sample of unknown volume is applied to the sample application site 110.
(2) As sample flows into the cartridge through passageway 120, it is detected through the sample detection window 122 using a light source and detector located in the monitor in which the cartridge is located (not shown).
(3) Sample continues to flow into the cartridge, filing passageway 120 to the rupture ~unction at 147.
The sample does not break the rupture ~unction 147 or ~low into rupture tank 130, since rupture tank vent 134 i5 closed (by the moni-tor).
(4) Sample continues to flow into the cartridge through passageway 120 and ~hrough control valve 125.
(5) Sample continue~ to flow into the cartridge and passa~eway 120, entering the measuring chamber at a junction between vertical segment 140a and hori~ontal segment 140b. Sample moves throu~h vertical segment 140a to upper stop-flow junction 146 and through horizontal segments 140b and 140c to lower stop-flow junction 145. Sample does not break lower stop-flow junction a~ 145 and flow into the dilution and mixing chamber 150 since valve 225 is closed and mixing chamber surge tank 154 is not vented to atmosphere (i.e., valve 225 and vent 156 were pre~iously closed by the monitor).
(6) Shortly after sample is detec~ed at upper stop-flow junction 146, rupture tank vent 134 is opened to atmosphere by the monitor.
(7~ As soon as rupture tank vent 134 is opened to atmosphere, control valve 125 is closed by the moni~or.
Since rupture junction 147 is designed to provide the least resistance to flow along the sample flow path, any 030191 50.

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shock that is created by closing conkrol valve 12S is ~9 dissipated by sample flowing into rupture tank 130.
This maintains the position of the sample at upper stop-flow junction 146 and lower stop-flow junction 145. By closing control valve 125, the portion of sample filling the capillary passageway at se~ments 140a, 140b, and 140c is isolated from the rest of the sample, which will remain in the various parts of passageway 120 during remaining operations, including the portion of passageway 120 to the right of valv0 125 but to the left of the junction of passageways 140a and 140b. The isolated porkion in measuring chamber 140 is a precise portion of the original sample.
(8) At this point, dilution and mixing chamber 150, the vent channel 153, and the mixing chamber surge tank 154 are opened to atmosphere by the monitor opening vent 156.
(9) Next, diluent ampule 175 i-~ broken by a blow on target point 278 (provided by the moni.tor), and diluent flows through the diluent application site 170 into channel 180 and fills channel 180 to and including vent 182.
(10) Once diluent fills the diluent application site 170 and ad~oining spaces, the additional hydrostatic pressure transmitted through sample in measuring chambers 140a, 140b, and 140c on the lower stop-flow junction 145 causes flow of diluent and the sample isolated in the measuring chamber 140 into mixing chamber 150. An initial segment 153a of channel 153 leading to stop-flow junction 152 is also filled by the diluted mixture.
(11) Once mixing chamber 150 has been completely filled, a mixing ball (not shown) in chamber 150 is reciprocated, mixing the diluent and sample, thsreby completing the first dilution sequence.
(12) To begin the second dilution, control valve 225 is opened by the monitor, allowing a portion of the 20~99751 030191 S1.

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mixture from mix:ing chamber 150 to flow into the rest of the cartridge.

(13) Sample (i.e., the fir~t mixture) flows through passageway 220 and valve 225 and into the upper arm 240a and lower arms 24Ob and 240c of the mixture measuring passageway 240. Flow stops at upper stop-flow junction 246 and lower stop-flow junction 245. Sample does not breaX lower stop-flow ~unction 245 and flow into khe the second mixing chamber (250) since ~ent 256 i~ closed by the monitor and surge tank 254 is not vented to atmosphere.

(14) Shortly after sample is detected by the monitor at upper stop-flow junction 246, control valve 225 is closed. By closing this walve khe portion o~
diluted sample (from the first dllution) Pilling mea~uring segments 240a, 240b, and 240c is isolated ~rom the rest of the sample. The isolated portion is a precise portion of the mixture from dilu-tion and mixing chamber 150.

(15) At this point, mixing chamber 250, vent channel 253, and surge tank 254 are opened to atmosphere by opening vent 256.

(16) Next, the diluent ampule 275 is broken, and diluent flows through diluent application site 270 and diluent channel 280, filling these chambers to vent 282.

(17) Once diluent iills dilluent channel 280, additional hydrostatic pressure on lower stop-flow junction 245 causes diluent and the sample in segments 240a and 240b of the measuxing chamber to flow into second mixing chamber 250, vent 252, and the portion of vent channel 253 below stop-flow junction 255.

(18) Once ~econd mixing chamber 250 has been completely filled, a mixing ball (not shown~ is reciprocated, mixing the diluent and sample and completing the dilution sequence.

202~9751 030191 52.

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(3) Chemical ~rocesses occuxrinq in the diluter Figure 8 i5 a schema1;ic diagram showing rea-gents that could be used with a cartridge of the type as shown in Figures 6 and 7 to carry out a specific diagnosis. A therapeutic drug/ such as theophylline, is assayed turbidimetrically in the cartridge by a latex agglutination-inhibition method. The a3say uses whole blood as the sample. To convert the sample to a form suitable for the as~ay, red ce:Lls and other "formed elements~ are dis301ved by dilution into a medium containing detergent. The assay system (monitor and reagent-containing cartridge) accomplishes this dissolution by a combined dilution/mixing step (the first dilution) and then performs a second dilution/mixing operakion to combine dilut~d sample with two initially dry reagents that are dissolved and resuspended by mechnical mixing. One of the reagents is a dispersion of latex particles which agglutinat0 at a rate inversely related to drug concentra~ion in the sample. The assay reaction is measured by following the increase of turbidity in the reaction medium over about 20 seconds.
A sample from an unmeasured blood drop will be applied to sample application site 110. Sample will flow into measuring chamber 140 through passageway 1~0.
When container 175 is broken, a detergent solution, which also contains excess ferricyanide and azide, will flow through measuring chamber 140 into mixing chamber 150, pushing the blood sample ahead of itself The mixture of blood and first reagent/diluent solution will fill mixing chamber 150. Homogeneous mixing of blood and the first diluent will now occur, driven by a reciprocating mixer. Ferricyanide converts the hemoglobin to it met form, which complexes with a~ide to give a well-defined spactral complex. The hemoglobin 0301gl 53.

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concentration of the sample is then calculated by measuring the absorbance of the diluted blood at 560 nm.
The plasma concentra~ion of drug can then be calculated from the concentration in the blood hemolysate from a simple mathematical relationship, no matter what the original sample hematocrit was.
The valve in channel 220 will then be opened to allow a portion of the mixture to flow into the measurement (mixture isolation) chamber system. Once the mixture measuring chamber 240 haq been filled, diluent container 275 is broken, allowing a glycine buffer diluent to flow into the dry antibody-latex reagent chamber 250 t resuspending the reagent (which is coated on the chamber walls of cha~er 250), after displacing the sample of denatured blood (i.e., the isolated mixture) from mixture measurement chamber 240 into the mixing/reaction chamber 250. Two dry reagen~s are present at different locations in chamber 250.
Reagent 1 contains drug-labelled latex particles and anti-mouse immunoglobulin ("second antibody"). Reagent 2 contains mouse monoclonal antibody to drug. The denatured blood/reagent mixture will then be mixed and assayed for theophylline by measurement of the change in turbidity over about 20 seconds. ~n absence of drug in the sample, the anti-drug binds to the drug-labeled latex particles. This is not enough to cause rapid agglutination of the latex. The second antibody binds to the anti-drug both free in solution and bound to the latex, thereby aggllltinating the particles. Drug in the sample competes with drug bound to latex for anti-drug antibody and thus inhibits the agglutination reaction.
The assay is set up ~o that over the clinically relevant range of drug, the agglutination reaction is almos~
fully inhibited.
The proper operation of a diluter using stop-flow ~unctions of the invention has been demon~trated using this chemistry. Other assays, such as those described in U.S. Application Serial No. 337,286t filed 202gg751 030191 54.

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All publications and patent applications mentioned in this specificatioll are herein incorporatad by reference to the same extent; as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one o~ ordinary skill in the art that many changes and modifical:ions can be made thereto without departing ~rom the spirit or ~cope of the appended claims.

030191 55.

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Claims (47)

1. In a device which comprises a capillary stop-flow junction located in a housing at an end of a capillary passageway used to transport a liquid and at the beginning of a non-capillary internal chamber in said housing, an improvement which comprises:
a. means for selectively trapping a gas in said capillary passageway and non-capillary chamber, wherein when said means for trapping is activated and said liquid enters said capillary passageway, said gas is compressed by said liquid as said liquid flows through said capillary channel and stops flowing at said stop-flow junction; or b. a stop-flow nozzle surrounding said capillary passageway and projecting into said chamber;
or c. said stop-flow junction being formed from a single housing body member; or d. a rupture junction in said capillary pathway, wherein said rupture junction is a stop-flow junction providing less maximum available back pressure than said capillary stop-flow junction.

2. The device of Claim 1, wherein said improvement comprises said means for selectively trapping gas.

3. The device of Claim 2, wherein said means for selectively trapping gas comprises a vent leading to atmosphere surrounding said device and means for selectively closing said vent.

4. The device of Claim 3, wherein (1) said vent is located in a housing containing said capillary passageway, said chamber, said stop-flow junction, and said vent and (2) said means for selectively closing 030191 56.

said vent comprises a sealing member located externally to said housing which is capable of selectively closing said vent.

5. The device of Claim 4, wherein said housing is adapted to removably fit into an electronic instrument and said sealing member is located in and operated by said electronic instrument.

6. The device of Claim 1, wherein said improvement comprises said stop-flow nozzle.

7. The device of Claim 6, wherein said nozzle comprises an opening where said capillary passageway enters said chamber and a surface adjacent to said opening and wherein said surface forms an acute angle with an adjacent surface of a wall of said capillary passageway.

8. The device of Claim 7, wherein said nozzle is formed in the shape of a coaxial cone surrounding said capillary passageway and projecting into said chamber.

9. The device of Claim 7, wherein said nozzle projects into a recess in a wall of said chamber, wherein said nozzle has a tip that does not extend out of said recess.

10. The device of Claim 1, wherein said stop-flow junction is formed Prom a single housing body member.

11. The device of Claim 1, wherein said improvement comprises said rupture junction.

12. The device of Claim 11, wherein said capillary passageway further comprises a valve located in said passageway between said capillary stop-flow junction and said rupture junction.

030191 57.

13. The device of Claim 12, wherein said valve comprises a flexible wall of said passageway which is biased to provide an open passageway when external forces are absent but which is capable of blocking said passageway when subjected to an externally applied force.

14. The device of Claim 11, wherein said rupture junction provides a back pressure at least 10% less than that of said capillary stop-flow junction.

15. The device of Claim 1, wherein said device comprises more than one of said improvements.

16. The device of Claim 1, wherein said device comprises at least three of said improvements.

17. The device of Claim 1, wherein said device comprises all of said improvements.

18. A vent-assisted capillary stop-flow junction, comprising in a housing surrounded by a gaseous atmosphere:
i. an internal chamber;
ii. liquid receiving means fox accepting a liquid;
iii. a capillary channel connecting said liquid receiving means to said chamber;
iv. a stop-flow junction at the intersection of said capillary channel and said chamber;
v. means for selectively trapping gas in said capillary channel and said chamber;
wherein when (1) said means for trapping is activated and gas is trapped in said capillary passageway and said chamber and (2) said liquid is concurrently applied to said means for accepting a liquid, said liquid flows through said capillary 030191 58.

channel, compresses air trapped in said housing by said means for trapping, and stops flowing at said stop-flow junction.

19. The vent-assisted stop-flow junction of Claim 18, wherein said means for trapping comprises a channel leading out of said chamber and connecting said chamber to the atmosphere surrounding said housing and means for selectively closing said channel and sealing said chamber from said atmosphere.

20. The vent-assisted stop-flow junction of Claim 19, wherein (1) said vent is located in a housing containing said capillary passageway, said chamber, said stop-flow junction, and said vent and (2) said means for selectively closing said vent comprises a sealing member located externally to said housing which is capable of selectively closing said vent.

21. The device of Claim 20, wherein said housing is adapted to removable fit into an electronic instrument and said sealing member is located in and operated by said electronic instrument.

22. A method for increasing stability of a capillary stop-flow junction in a housing surrounded by a gaseous atmosphere, wherein said housing contains:
i. an internal chamber of non-capillary dimensions;
ii. liquid receiving means for accepting a liquid;
iii. a capillary channel connecting said sample receiving mean to said chamber;
iv. said-stop-flow junction at the intersection of said capillary channel and said chamber; and 030191 59.

v. a venting channel leading out of said chamber and connecting said chamber to the atmosphere surrounding said housing;
which comprises the step of:
closing said venting channel prior to applying said liquid to said means for accepting a liquid, whereby said liquid flows through said capillary channel and stops flowing at said stop-flow junction.

23. The method of Claim 22, wherein said venting channel has a volume selected 50 that compression of air trapped in said capillary channel, said chamber, and said venting channel produces an internal pressure that opposes flow of said liquid that is no greater than hydrostatic pressure acting in the direction of said flow.

24. A rupture-junction stabilized stop-flow junction, which comprises in a housing:
i. an internal chamber;
ii. liquid receiving means for accepting a liquid;
iii. a capillary channel connecting said liquid receiving means to said chamber;
and iv. a first stop-flow junction at the intersection of aid capillary channel and said chamber; and v. a rupture junction in said capillary passageway consisting of a second stop-flow junction leading to a second internal chamber, wherein said second stop-flow junction has a maximum available back pressure less than that of said first stop-flow junction.

25. The rupture-junction stabilized stop-flow junction of Claim 24, wherein said second stop-flow junction has 030191 60.

a mazimum available back pressure at least 10% less than that of said first stop-flow junction.

26. The rupture-junction stabilized stop-flow junction of Claim 24, wherein a valve is present in said capillary passageway between said first and said second stop-flow junctions.

27. A method for designing device comprising a vent-assisted stop-flow junction in a housing surrounded by a gaseous atmosphere, wherein said housing contains:
i. an internal chamber;
ii. liquid receiving means for accepting a liquid;
iii. a capillary channel connecting said sample receiving means to said chamber;
iv. said stop-flow junction at the intersection of said capillary channel and said chamber; and v. a venting channel leading out of said chamber and connecting said chamber to the atmosphere surrounding said housing;
which comprises:
selecting internal volumes in said device and providing one or more externally controlled vents so that compression of trapped gas that would otherwise escape through said vent or vents takes place in an enclosed space created by closing all vents to that space with the advancing liquid supplying the compressive force, with the resulting increase in internal gas pressure being used to oppose the flow of sample past the stop-flow junction under consideration.

28. An apparatus for automatically carrying out a dilution of a sample with a diluent in a housing, comprising in said housing:
(1) a sample application site for receiving a sample;

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(2) a rupture chamber comprising a vented interior chamber;
(3) a mixing chamber comprising a vented interior chamber having a first volume;
(4) a diluent application site for receiving a diluent;
(5) capillary flow means comprising:
(a) a valved segment having a first and a second end;
(b) a valve located in said valved segment;
(c) a sample segment connecting said sample application site to said first end of said valved segment;
(d) a rupture segment connecting said rupture chamber to said first end of said valved segment;
and (e) a measuring segment connected to said second end of said valved segment and having first and second exits t wherein said first exit connects said measuring segment to said mixing chamber and wherein said measuring segment has a second volume smaller than said first volume of said mixing chamber;
(f) a first stop-flow junction located at said first exit of said measuring segment and adapted to the surface-tension characteristics of the sample so as to provide sufficient back pressure resulting from contact between the sample and wall means of said housing at said first stop-flow junction to prevent sample from flowing through said first stop-flow junction in the absence of diluent;
(g) a second-stop flow junction located at said second exit of said measuring segment and adapted to the surface-tension characteristics of the sample so as to provide sufficient back pressure resulting from contact between the sample and wall means of said housing at said second stop flow junction to prevent sample from flowing 030191 62.

through said second stop-flow junction in the absence of diluent;
(h) a third stop-flow junction located at the junction of said rupture segment and said rupture chamber and adapted to the surface-tension characteristics of the sample 50 as to provide sufficient back pressure resulting from contact between said sample and wall means of said housing at said third stop flow junction to prevent sample from flowing through said third stop-flow junction in the absence of diluent, wherein said third stop-flow junction provides less back pressure than said first stop-flow junction;
whereby addition of sample to said sample application site causes sample to fill said capillary flow means;
(6) diluent flow means connecting said diluent application site to said first exit of said measuring segment.

29. The apparatus of Claim 28, further comprising a mixture isolation chamber connected to said mixing chamber by a flow means for delivering a portion of the contents of said mixing chamber to said mixture isolating chamber by the sum of capillary and gravitational forces; and normally closed second valve means selectively preventing flow between said mixing chamber and said mixture isolating chamber, whereby opening said second valve means causes a measured representative sample of liquid in said mixing chamber to flow into said isolating chamber.

30. The apparatus of Claim 28, further comprising means for selectively trapping gas in an interior space formed in part by at least a portion of said capillary passageway.

030191 63.

31. The apparatus of Claim 30, wherein said means for selectively trapping gas comprises (1) a vent connected to said portion of said capillary passageway either directly or by means of interior spaces in said housing which lead to atmosphere surrounding said apparatus and (2) means for selectively closing said vent.

32. The apparatus of Claim 31, wherein (1) said vent is located in a housing containing said capillary passageway, said chamber, said stop-flow junction, and said vent and (2) said means for selectively closing said vent comprises a sealing member located externally to said housing which is capable of selectively closing said vent.

33. The apparatus of Claim 32, wherein said housing is adapted to removable fit into an electronic instrument and said sealing member is located in and operated by said electronic instrument.

34. The apparatus of Claim 28, wherein at least one stop-flow junction in said apparatus comprises a nozzle projecting into said chamber.

35. The apparatus of Claim 34, wherein said nozzle comprises an opening where said capillary passageway enters said chamber and a surface adjacent to said opening and wherein said surface forms an acute angle with an adjacent surface of a wall of said capillary passageway.

36. The apparatus of Claim 35, wherein said nozzle is formed in the shape of a coaxial cone surrounding said capillary passageway and projecting into said chamber.

37. The apparatus of Claim 36, wherein at least one stop-flow junction in said apparatus is formed from a single housing body member.

030191 64.

38. The apparatus of Claim 34, wherein at least one stop-flow junction in said apparatus is formed from a single housing body member.

39. The apparatus of Claim 28, further comprising a valve in said capillary flow means.

40. The apparatus of Claim 29, wherein said valve comprises a flexible wall of said passageway which is biased to provide an open passageway when external forces are absent but which is capable of blocking said passageway when subjected to an externally applied force.

41. The apparatus of Claim 40, wherein said third stop-flow junction provides a back pressure at least 20%
less than that of said capillary stop-flow junction.

42. The apparatus of Claim 28, wherein said diluent is retained in a frangible container enclosed in an internal vented diluent chamber in said housing, wherein said diluent housing further comprises said diluent application site.

43. The apparatus of Claim 42, wherein said frangible container is adapted to fit closely within said said diluent chamber, wherein a blow to a wall of said housing at said diluent chamber is capable of deforming said wall and breaking said container.

44. The apparatus of Claim 43, wherein a portion of said housing at said wall is thinner than adjacent portions of said housing, thereby providing a flexible target region.

030191 55.

45. The apparatus of Claim 44, wherein said target region comprises a central thick target point surrounded by thinner housing.

46. The apparatus of Claim 28, wherein at least one vent channel leading from an interior channel or chamber designed to contain liquid during a measurement or dilution step enters an excess liquid holding tank in the interior of said housing of sufficient size to retain said liquid, with an external vent to said external atmosphere being provided at a location in said holding tank not reachable by said liquid when said apparatus is properly oriented.

47. The apparatus of Claim 46, wherein said external vent also forms part of a vent-assisted stop-flow system.

030191 66.