Use of fluid retarding ion conducting material

An improved particle sensing transducer apparatus and method for studying the physical properties of particles suspended in an electrolyte solution. The transducer apparatus includes a first chamber, at least a portion of which contains a quantity of the electrolyte solution with a first electrode disposed therein. A second chamber is provided, at least a portion of which contains a quantity of the electrolyte solution with a second electrode disposed therein. The transducer further includes an orifice for establishing a constricted electrical path by providing a passageway for a sample flow of electrolyte solution containing the particles between the two chambers. The improvement comprises a fluid retarding, ion conducting material, such as a gel, frit or membrane, interposed between the sample flow and at least one of the electrodes so as to pass an ionic current while retarding the electrolyte flow from the electrode.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to the use of fluid retarding, ion conducting 
material used to isolate electrolyte products from the region of 
electronic sensing of particles. 
2. Discussion of the Prior Art 
Various impedance or phase based particle sensing devices exist in the 
prior art for studying the physical properties of microscopic particles, 
such as biological cells carried in a liquid suspension, as illustrated by 
the pioneer U.S. Pat. No. 2,656,508, "Means for Counting Particles 
Suspended in a Fluid" W. H. Coulter, Oct. 20, 1953; and U.S. Pat. No. 
4,014,611, "Aperture Module for Use in Particle Testing Apparatus", 
Simpson et al., Mar. 29, 1977. A well known "Coulter principle" of 
operation is referred to with particularity in these patents. Generally, 
these Coulter devices include two fluid vessels or chambers, each 
containing a conductive electrolyte solution. At least two electrodes 
having opposite polarity are immersed in the electrolyte solution, with 
each fluid compartment having one of the electrodes disposed therein. A 
sample of the electrolyte solution, having the particles suspended 
therein, is passed through a constricted fluid path or orifice interposed 
between the two fluid compartments. Although this constricted path can 
take different forms, in each device such path defines a sensing zone 
wherein the presence or absence of a particle in the constricted path 
gives rise to a detectable change in electrical characteristics of the 
path. For example, relatively poorly conductive biological cells passing 
through this constricted path displace a volume of electrolyte solution 
equal to the cell volume, causing a voltage drop by increasing the path 
impedance. To put it another way, the resistance between the two 
electrodes which are separated by the constricted path is increased by the 
cell presence. The resistance pulses defined by the voltage drops are used 
for particle counting and particle volume determination. 
Modification of the above described prior art sensing scheme has led to the 
development of particle sorters, wherein the selective resistance pulses 
provided by constricted path activates the sorter to charge individually 
isolated droplets containing the activating cells. The charged droplets 
are deflected from the main stream by a static electric field into a 
collecting vessel. Typically, the prior art sorters include a first and a 
second sheath flow, with the second sheath flow being introduced below the 
orifice. A downstream return electrode is mounted in the second sheath. 
Consequently, the downstream particles are exposed to undesirable 
electrode products produced by the return electrode. An illustrative 
particle sorter has been sold by Coulter Electronics, Inc. of Hialeah, 
Florida. 
In order to sense the impedance changes, it is necessary to have a current 
flow between at least two electrodes in the case of DC currents. The 
current flow is due to ions which proceed toward the oppositely charged 
electrode. However, there are several inherent problems brought about by 
this electrolysis process, which next will be discussed. 
Almost all electrolyte solutions create unwanted gas at the electrodes. For 
instance, the electrolyte sodium chloride in solution (saline solution) 
forms oxygen, chlorine and hydrogen gases which take the form of gas 
bubbles, such bubbles frequently create noise in the impedance sensing 
device as such bubbles travel through the constricted path. At the same 
time, other undesirable electrolyte products are produced. For example, in 
the case of sodium chloride, hypochlorite is formed by the chlorine gas 
acting with the water, and can kill or damage biological cells. 
Electrolysis normally changes the pH of the solution, such as where 
hydrogen ions form hydrogen and thereby make the solution more basic. 
Cells generally are viable only in a specific pH range, and such pH 
changes can even kill the cells. Moreover, the user may be operating the 
impedance sensing device based on assumed cell environment conditions. 
However, a change in pH, and therefore a change in cell environment, can 
lead to different physical properties of the cell, such as changes in the 
cell membrane. These different physical properties can lead to a change in 
cell volume; hence, a change in the detected resistance. Moreover, the 
electrodes can be fouled by the presence of various substances, including 
proteins. 
Accordingly, it readily can be seen that there has been a long recognized 
need in the art of cytology to prevent the electrolyte products from 
interfering with the impedance sensing device and sorting processes. 
In the case of simple impedance based cell sorters, such as the previously 
cited sorter, or more simply where the cells are to be collected, it is 
necessary to minimize the volume of liquid beneath the orifice. First, 
this minimized volume is desirable for the purpose of providing fidelity 
of collection, and secondly, not impeding fluid flow. Since the power 
electrodes must be of a finite size, it is necessary to position the 
downstream electrode remotely from the orifice. 
The use of frits, gels and membranes in chemical art areas is well known. 
For instance, electrophoresis involves the movement of charged, dispersed 
particles in a colloidal system toward electrodes that have opposite 
charges, such process normally being used to separate molecular species, 
such as proteins which differ by charge or charge and shape. In order to 
separate properly the molecular species, it is desirable not to have 
bubbles which create fluid turbulences and changes in pH, which effect the 
mobility (velocity) of the species being separated. In short, a constant 
chemical composition of the solution employed to perform these separating 
tests is required. Consequently, fruits and other such means are used to 
separate the volume holding the electrodes from the volume in which 
separation occurs. However, there is no electronic sensing of individual 
particles in the electrophoresis process. 
In prior art pH and other ion sensing meters, frits, gels and like means 
are used to protect and separate and maintain the precisely defined 
chemical milieu that is disposed around the internal electrode of the 
reference electrode from the solution being measured by the pH meter. 
However, in that there is a minimal amount of current in the pH meters, 
electrolyte products are of negligible consequence. The current in a pH 
meter is of the order of one billionith of that in a standard particle 
sensing transducer. Other chemical apparatuses, such as polargraphs and 
electrolylic half cells, use various conducting gels and frits. However, 
none of these processes involve impedance sensing of particles. 
SUMMARY OF THE INVENTION 
The present invention relates to an improved particle sensing transducer 
apparatus and method for studying the physical properties of particles 
suspended in an electrolyte solution. The sensing transducer is of the 
type using the "Coulter principle" of operation, wherein there is provided 
an orifice which forms a constricted path for a sample flow of electrolyte 
solution having a quantity of the particles suspended therein. The orifice 
also defines a constricted electrical path for an ionic current provided 
by a pair of electrodes, such electrodes being fluidly disposed on 
opposite sides of the orifice. The improvement comprises interposing a 
fluid retarding, ion conducting material between at least one of the pair 
of electrodes and the sample flow, thereby substantially isolating the 
sample flow from disruptive and harmful electrolyte products. These 
electrolyte products can include gas bubbles, which are formed at the 
electrode disposed upstream with respect to the orifice, that can pass 
through the orifice and produce inaccurate impedance readings. Also, these 
electrolyte products may include various noxious substances generated by 
the electrodes which damage the particles. Depending upon the application 
for which the sensing transducer apparatus is used, the present invention 
contemplates protecting the sample flow from the noxious substances 
produced by the electrode disposed either downstream or upstream with 
respect to the orifice, or by both electrodes. Moreover, the downstream 
use of a fluid retarding, ion conducting material allows for the 
minimizing of the volume of liquid beneath the orifice, and thereby is 
advantageous in those applications wherein the particles are to be 
collected.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
A particle sensing transducer, generally identified as 10, is illustrated 
in FIG. 1. There is provided a first chamber 12 in the form of a tube 
which holds an electrolyte solution. A second chamber 14 is arranged with 
the first chamber 12 positioned therein; and the second chamber 14 also is 
provided with a quantity of electrolyte solution. Near the bottom of the 
first chamber 12 and in its side wall there is provided an orifice 16. The 
electrolyte solution is caused to flow from the second chamber 14 through 
the orifice 16 and into the first chamber 12 by virtue of an external 
vacuum source 18 (partially shown). A dilute suspension of particles, such 
as biological cells, is contained in the electrolyte solution of the 
second chamber 14. A stopcock 19 is opened and the external vacuum source 
18 initiates a sample flow of electrolyte solution, having a quantity of 
the particles suspended therein, through the orifice. The sample flow is 
defined as that fluid which passes through the orifice. A pair of 
electrodes, a first electrode 20 and a second electrode 22, is immersed in 
the electrolyte solution. More specifically, the first electrode 20 is 
inserted in the first chamber 12 and the second electrode is inserted in 
the second chamber 14. By virtue of ion conduction, an electrical current 
is arranged to flow between the electrodes through the orifice 16, so that 
the presence of a particle in the orifice 16 causes a change in current 
flow. The orifice 16 forms a constricted electrical path which defines a 
sensing zone 23 for the detection of impedance changes caused by the 
particles. The electrodes 20 and 22 are attached to a detecting device 24 
by leads 25 and 26. 
The above described transducer structure is well known in the art and can 
take many different conventional forms, all of which can make use of the 
invention to be described hereinafter. In general, these particle sensing 
transducers of the prior art have two electrolyte chambers, such as the 
first chamber 12 and second chamber 14, which are interconnected by a 
constricted fluid path, such as the orifice 16. Particles are introduced 
into one of the chambers and a sample flow having the particles suspended 
therein is drawn through the constricted path into the other chamber. The 
specific structure of this arrangement per se forms no part of the present 
invention. 
It should also be appreciated that in the prior art impedance sensing 
transducers, the second electrode 22 would have been inserted directly 
into the second chamber 14. Consequently, the particles would be exposed 
to the previously described harmful electrolyte products prior to their 
passage through the orifice 16. Moreover, gas bubbles formed at the second 
electrode 22 could pass with the sample flow through the orifice 16 and 
create the previously described, inaccurate impedance readings. 
The embodiment of the present invention illustrated in FIG. 1 contemplates 
isolating the second electrode 22 from the sample flow by use of a filter 
means in the form of a gel 28, which is interposed between the second 
electrode 22 and the orifice 16. This is contrary to the prior art 
practice which would normally involve inserting the electrode 22 into the 
chamber containing the particles. More specifically, one way to implement 
this separation is to divide the second chamber 14 into two compartments 
29 and 30, each of which holds a quantity of the electrolyte solution. 
This can be accomplished by the insertion of a dividing wall 31 into the 
second chamber 14 as illustrated in FIG. 1, or by other obvious 
adaptations, such as having two spaced-apart compartments. In compartment 
29, the sample flow, having the particles, is moved to the orifice 16; 
whereas, the compartment 30 has the second electrode 22 disposed therein. 
The compartment 29 is connected electrically to the compartment 30 by 
means of a passageway 32, such passageway encasing the gel 28. By virtue 
of this arrangement, ion conduction can proceed between the electrodes 20 
and 22 by passing through the gel 28. On the other hand, fluid flow from 
the compartment 30 is retarded by the gel 28. By virtue of this electrical 
conduction, fluid retarding relationship, the particles in the compartment 
29 are not exposed to noxious electrode products produced at the second 
electrode 22, such as chlorine gas, hypochlorite, and changes in pH, prior 
to their passing through the orifice 16. Moreover, bubbles formed at the 
second electrode 22 do not pass through the gel 28 and therefore do not 
pass through the orifice 16. Since the sample flow proceeds into the first 
chamber 12, bubbles and electrolyte products produced at the first 
electrode 20 do not normally affect the particles until after they enter 
the first chamber 12 and pass through the sensing zone 23 of the orifice 
16. Moreover, the fluid flow through the orifice 16 prevents any bubbles 
formed at the first electrode 20 from entering the sensing zone 23 of the 
orifice 16. 
It should be appreciated that materials and means other than gel 28 can be 
used for the filter means to provide an electrical impedance which is 
relatively small, while retarding or stopping the fluid flow. For example, 
as illustrated in FIG. 2, a frit or a membrane or like materials can be 
used in place of or in addition to the gel 28 and is arranged on the 
assumption that the hydrostatic force is exerted into the frit containing 
end of the passageway 32. Such materials can be used singly or in 
combination. More specifically, gels are less permeable to fluid, while 
frits provide more structural support, but generally are more permeable to 
fluid. Where there is a substantial hydrostatic pressure, it may be 
desirable to use the frit 34 and the gel 36. Moreover, as shown in the 
embodiment of FIG. 2, if the hydrostatic pressure is great enough, it may 
be desirable to use two types of gels. The first type of gel preferably 
should be rigid, such as a cross-linked gel 36, which is located next to 
the frit 34, and the second gel is a conventional gel 38. The cross-linked 
gel 36 is more resistant to deterioration under hydrostatic pressure and 
thus can act as a plug to stop the conventional gel from being pushed 
through the frit 34. Generally, the conventional gel has a somewhat faster 
diffusion rate for the ions than the cross-linked gel, but the 
cross-linked gel provides more resistance against hydrostatic pressure. In 
FIG. 1, only the gel 28, possibly an agar gel, is shown in the passageway 
32. The use of only the gel 28 is sufficient in such an application as 
that illustrated in FIG. 1 in that there is practically no hydrostatic 
pressure. However, as illustrated in FIG. 2, frit 34 and/or a combination 
of the cross-linked gel 36 and conventional gel 38 can be included where 
significant hydrostatic pressures exist. The cross-linked gel 36 can be a 
commercially available gel which is cross-linked, for example by 
glutaraldehyde fixation, a well known cross-linking agent, to immobilize 
the same within the passageway 32. By virtue of this arrangement, the 
molecules of the cross-linked gel 36 are formed into a three dimensional 
matrix which is sufficiently rigid to prevent the hydrostatic pressure 
from pushing the conventional gel through the porous frit 34. It should be 
understood that frits and gels are merely used as illustrative examples 
and that any material, such as ultrafiltration membranes commercially 
available from Amnicon Corp. of Lexington, Massachusetts, which 
sufficiently retards fluid flow while allowing ion flow is within the 
scope of this invention. A membrane which could be useful in this 
implementation is a thin substrate that impedes the flow of fluid, yet 
permits the flow of ions, such as a thin gel pulled taut over a supporting 
structure. Also, such flow retarding materials do not have to stop all of 
the fluid flow, in that some fluid flow through the flow retarding 
material may be acceptable for a particular application. 
Another possible application of the fluid retarding, ion conducting 
material can be understood by initially referring back to FIG. 1. As 
previously described, the particles are not exposed to the electrolyte 
products from the second electrode 22 before passing through the sensing 
zone 23. As shown in FIG. 1, the particles would, after passing through 
the sensing zone 23, be exposed to electrolyte products from the first 
electrode 20. It may be desirable to collect viable cells and/or avoid 
undesirable buildup of electrolyte products on the first electrode 20. To 
avoid these undesirable features of electrolysis, the first electrode 20 
also can be fluidly isolated by a gel or like means in a manner similar to 
that of the second electrode 22. Such an implementation is illustrated in 
FIG. 3. 
Referring to the cross-sectional view of FIG. 3, a particle sensing 
transducer 10 is illustrated with a first and a second chamber 12 and 14, 
respectively, such chambers being electrically interconnected through an 
orifice 44. The upstream second chamber 14 is divided into two electrolyte 
containing compartments, a first compartment 46 having a first electrode 
48 disposed therein and a second compartment 50 which is in fluid 
communication with the orifice 44. The first compartment 46 is 
electrically connected to the second compartment 50 by frit 52 and gel 54. 
The downstream first chamber 12 also is divided into two electrolyte 
containing compartments, a third compartment 56 having a second electrode 
58 therein and fourth compartment 60, which is in fluid communication with 
the orifice 44. The second electrode 58 preferably has a large surface 
area and can have, for instance, a circular configuration. The fourth 
compartment 60 is configured and dimensioned to receive a sample flow 
having the particles suspended therein, which passes from the second 
compartment 50, through the orifice 44, and into the fourth compartment 
60. The third compartment 56 and the fourth compartment 60 are 
electrically connected through a gel 62 and a cylindrically-shaped frit 
64. By virtue of this arrangement, close electrical contact can be made 
with the downstream fluid area, the fourth compartment 60, while 
preventing the electrode products from invading such area. 
Various well known downstream activities can occur with the above described 
structure of FIG. 3. For instance, particle sorting may be performed or, 
alternatively, the close electrical contact of the gel 62 and frit 64 can 
be part of an electrical arm in a bridge circuit. The embodiment of FIG. 3 
is intended to be generic in concept of all conventional particle sensing 
transducers, wherein downstream activities occur after the sample flow 
passes through the orifice 44. In these conventional transducers, it is 
desirable to minimize the length of fluid travel of the particles past the 
orifice 44. For example, the ability for particle sorting may be 
diminished or lost after a lengthy downstream fluid flow by the pressure 
of the fluid, delay in sorting, and jitter problems caused by the fluid 
flow. These problems can be decreased by the positioning of a downstream 
electrode immediately below the orifice 44. Although this reduces the 
fluid travel, as a practical matter, it results in the surface area of 
such electrode being minimized. A small surface area for the electrode in 
turn causes a new set of problems in the form of noise generated and 
overvoltage problems being maximized. The prior art has not been able to 
solve this dilemma and has resorted to using a second sheath for remotely 
disposing a large electrode. Although this allows for a large downstream 
electrode, the inclusion of the second sheath causes turbulances in the 
downstream fluid flow, so as to make control of particle positioning in 
the stream more difficult. Also, the second sheath uses large quantities 
of liquid, which is expensive. 
In FIG. 3, an access channel 66 provides for a relatively small electrical 
contact area with the downstream area of the fourth compartment 60, 
thereby minimizing the length of the downstream fluid flow. Additionally, 
the access channel 66 provides for the remote disposition of the electrode 
58. Hence, a sizable electrode surface may be provided which minimizes 
noise and overvoltage, without the use of the second sheath and its 
associated problems. 
A specific application of the fluid retarding, ion conducting material to a 
conventional particle sorting transducer 68, sold by Coulter Electronics, 
Inc. of Hialeah, Florida and identified in the Background Section, is 
illustrated in FIG. 4. The particle sorter itself is of conventional 
design and, for that reason, only the part which is modified by the 
present invention is illustrated in FIG. 4. More specifically, the 
conventional particle sorter normally comprises a sample flow of suspended 
particles which are ejected through a capillary 70. This sample flow is 
surrounded by a sheath flow and proceeds through the orifice 72. In this 
well known type of system, the sample flow or sample stream comprises a 
suspension fluid containing the particles, which flows down the capillary 
70. This sample flow is entrained by the sheath flow comprising sheath 
fluid (usually saline) which flows down the annular region between the 
capillary 70 and an inner wall 71. The combined flows laminarly proceed 
down to and through the orifice 72. The orifice 72 defines a sensing zone 
74 for receiving the flow sample. A pair of power electrodes, first and 
second electrodes 76 and 78, are in electrical communication with opposite 
sides of the orifice 72 so as to provide for an ionic current through the 
orifice 72. In practice, more electrodes can be involved in the detection 
of the particles. After proceeding through the sensing zone 74, liquid 
droplets containing particles are formed from the sample flow, which is in 
the form of a liquid jet, by applying to it small mechanical disturbances 
with ultrasonic frequencies. Thus, impedance sensing occurs, then a 
plurality of droplets are formed. Droplets containing cells to be sorted 
are charged and deflected from the main stream by a static electric field 
into a collecting vessel, in one implementation. All of this structure of 
the cell sorter is well known in the art. 
In the conventional particle sorting transducer of the prior art, as 
illustrated by the one identified in the Background Section, a second 
sheath flow is positioned below the orifice 72. Normally, a downstream 
electrode is positioned in the extremity regions of the second sheath. The 
electrode is normally held at ground potential to prevent the droplet 
charging pulses from entering the sensing zone 74. Referring to FIG. 4, 
this basic prior art scheme is modified by the present invention by 
eliminating the second sheath flow of the prior art and substituting 
therefor a gel 80 within a passageway 82. In this manner, electrical 
contact is made through the gel 80 or like material, instead of through 
the second sheath flow; while the first electrode 76 is isolated fluidly 
from the sample flow. In that a power electrode, such as first electrode 
76, requires an electrolyte solution to carry out the electrolysis 
process, the second electrode 78 is immersed in electrolyte solution 
contained in a fluid container 84. As with the other applications of a 
fluid retarding, ion conducting material heretofor described, frits (not 
shown) could be incorporated into the design for added mechanical 
strength, such frits being located in the passageway 82 to retain the gel 
80 against a hydrostatic force. 
In sorting cells, it is particularily important that the cells are not 
damaged by the electrolyte products; and, in some cases, it is desirable 
to have viable cells after sorting. Hence, not only is it necessary to 
prevent exposure of the cells to harmful electrolyte products before 
passing through the sensing zone 74, but exposure after passing through 
the sensing zone 74 must be avoided. Therefore, as illustrated in FIG. 4, 
the sample flow that has passed through the sensing zone 74 is protected 
from electrolyte products generated by the first electrode 76. With the 
second sheath of the prior art design, there was no such protection. 
Moreover, electrolyte solution can be expensive and, with the prior art 
design, large amounts of electrolyte solution were required to maintain 
the second sheath flow. 
It now can be appreciated that the same structural features of the 
invention exist in the embodiments of FIGS. 1, 3 and 4, even though the 
embodiment of FIG. 4 has been modified to a sheath flow system which is 
coupled into a particle cell sorting system. More specifically, a first 
chamber for electrolyte solution is disposed downstream relative to the 
orifice 72 and would include a liquid jet receiving area, generally 
indicated as 86, the passageway 82, and the container 84. Consequently, 
this first chamber comprises the receiving area 86 which defines a first 
fluid compartment; and the container 84 which defines a second fluid 
compartment; with the first and second fluid compartments being separated 
by the gel filled passageway 82. The first fluid compartment is in fluid 
communication with the orifice 72; while, the second fluid compartment has 
the first electrode 76 disposed therein. A second chamber is disposed 
upstream with respect to the orifice 72 and has the second electrode 78 
mounted therein and encompasses the sheath flow. 
In summary, it can be seen that there is a need for preventing exposure of 
particles to electrolyte products prior to passing the particles through 
an impedance sensing zone. As shown in FIG. 1, the electrolyte products 
from the upstream electrode (relative to orifice 16), the first electrode 
20, are blocked substantially by the gel 28 from coming into contact with 
the particles. This also prevents bubbles from entering the sensing zone 
23. It also can be seen that in some applications, such as cell sorting, 
or any other means of cell collection, there is a need for preventing 
exposure of particles to electrolyte products after the particles pass 
through the sensing zone. As illustrated in FIG. 4, the electrolyte 
products from the downstream electrode 76 (relative to orifice 72) are 
blocked substantially by the gel 80 from coming into contact with the 
particles. Yet, in both embodiments of FIGS. 1 and 4, the gel 28 or 80 
allows for the flow of ionic current. The use of the gel permits the 
downstream electrode to be remotely disposed frm the downstream flow and 
thus permits the geometry of this region to be optimized for cell sorting 
or other means of cell collection. 
Yet another application of the present invention is to utilize the fluid 
retarding, ion conducting material in a conventional particle sensing 
transducer, having a conventional bridge circuit (not shown), for 
measuring impedance in the sensing zone. An illustrative bridge circuit is 
disclosed in two articles in "The Journal of Histochemistry and 
Cytochemistry", by the Histochemical Society, Inc. The first article 
appears in Volume 22, No. 7, pp. 626-641, 1974 and is entitled 
"Computer-Based Electronic Cell Volume Analysis with AMAC II Transducer" 
and the other article appears in Volume 1, January, 1978 and is entitled 
"The AMAC IIA, A True Bridge Circuit Coulter-Type Electronic Cell Volume 
Transducer". It should be understood that the specific structure of the 
bridge circuit is not part of the present invention. These conventional 
bridge circuits normally have a series of connecting channels comprising 
small holes which connect the downstream sample flow with a displacement 
rheostat and a remote downstream power electrode. In addition to the 
previously described problems with downstream power electrodes, this 
bridge arrangement creates an additional problem if a fluid flow is used 
as the conductive element in these connecting channels. This fluid flow, 
when proceeding through the small channels, creates noise that interferes 
with the particle sensing. The replacement of this fluid flow with a fluid 
retarding, ion conducting material, such as various combinations of frits 
and gels, eliminates this source of noise. 
Although particular embodiments of the invention have been shown and 
described here, there is no intention thereby to limit the invention to 
the details of such embodiments. On the contrary, the intention is to 
cover all modifications, alternatives, embodiments, usages and equivalents 
of the subject invention as fall within the spirit and scope of the 
invention, specification and the appended claims.