Microlithographic array for macromolecule and cell fractionation

A sorting apparatus and method for fractionating and simultaneously viewing individual microstructures, such as free cells, viruses, macromolecules, or minute particles in a fluid medium. The sorting apparatus is composed of a substrate having a receptacle located therein, the receptacle having sidewalls and a floor. An array of obstacles is positioned within the receptacle with the obstacles upstanding from the floor of the receptacle. A transparent cover overlies the array of obstacles to cover the receptacle and afford visual observation of migration of the microstructures exclusively through the array of obstacles. Electrodes may be positioned within the receptacle to generate an electric field in the fluid medium in the receptacle in order to induce the migration of the microstructures. Migration of the microstructures may also occur, for example, by a hydrodynamic field, an optical field, a magnetic field, or a gravity field applied to the receptacle. The obstacles of the array of obstacles may be of various shapes such as round posts, rectangular bunkers, or v-shaped or cup-shaped structures. The arrays of obstacles are formed of a predetermined and reproducible pattern, and can be reused. Methods for manufacturing and using the apparatus are also claimed.

BACKGROUND 
1. The Field of the Invention 
The invention relates to apparatus and methods for fractionating 
microstructures such as free cells, viruses, macromolecules, or minute 
particles. More particularly, the present invention relates to apparatus 
and methods for sorting such microstructures in suspension in a fluid 
medium while simultaneously viewing individual of those microstructures 
during the process. 
2. Background Art 
The sizing, separation, and study of microstructures such as free cells, 
viruses, macromolecules, and minute particles are important tools in 
molecular biology. For example, this fractionation process when applied to 
DNA molecules is useful in the study of genes and ultimately in planning 
and the implementation of genetic engineering processes. The fractionation 
of larger microstructures, such as mammalian cells, promises to afford 
cell biologists new insights into the functioning of these basic building 
blocks of living creatures. 
A. Macromolecule Fractionation 
While many types of macromolecules may be fractionated by the apparatus of 
the present invention, the fractionation of a DNA molecule will be 
discussed below in detail as one example. 
The DNA molecules in a single cell of a complex organism contain all of the 
information required to replicate that cell and the organism of which it 
is a part. A DNA molecule is a double helical chain of four different 
subunits that occur in a genetically coded succession along the chain. The 
four subunits are the nitrogenous bases, adenine, cytosine, guanine, and 
thymine. The size of such a molecule is measured by the number of such 
bases it contains. Natural DNA molecules range in size from a few 
kilobasepairs in length to hundreds of megabasepairs in length. The size 
of a DNA molecule is roughly proportional to the number of genes the 
molecule contains. 
The size of a DNA molecule can also be expressed by its molecular weight, 
its length, or the number of basepairs it includes. If the number of 
basepairs is known, that number can be converted into both the length and 
the molecular weight of the DNA molecule. One method for estimating the 
size of small DNA molecules is the process of gel electrophoresis. 
In gel electrophoresis an agarose gel is spread in a thin layer and allowed 
to harden into a firm composition. The composition comprises a fine 
network of fibers retaining therewithin a liquid medium, such as water. 
The fibers of the agarose gel cross and interact with each other to form a 
lattice of pores through which molecules smaller than the pores may 
migrate in the liquid retained in the composition. The size of the pores 
in the lattice is determined generally by the concentration of the gel 
used. 
Slots are cast in one end of the gel after the gel is hardened, and DNA 
molecules are placed into the slots. A weak electric field of typically 
1-10 volts per centimeter is then generated in the gel by placing the 
positive pole of an electric power source in one end of the gel and the 
negative pole of the power source in the opposite end. In DNA 
electrophoresis, the negative pole of the power source is placed in the 
gel at the end of the composition in which the slots containing the DNA 
are located. The DNA molecules, being negatively charged, are induced by 
the electric field to migrate through the gel to the positive pole of the 
power source at the other end of the composition. This occurs at speeds of 
typically only a few centimeters per hour. 
The electrophoretic mobility of the molecules can be quantified. The 
electrophoretic mobility of a molecule is the ratio of the velocity of the 
molecule to the intensity of the applied electric field. In a free 
solution, the mobility of a DNA molecule is independent of the length of 
the molecule or of the size of the applied electric field. In a hindered 
environment, however, aside from the structure of the hindered 
environment, the mobility of a molecule becomes a function of the length 
of the molecule and the intensity of the electric field. 
The gels used in gel electrophoresis is just such a hindered environment. 
Molecules are hindered in their migration through the liquid medium in the 
gel by the lattice structure formed of the fibers in the gel. The 
molecules nevertheless when induced by the electric field, move through 
the gel by migrating through the pores of the lattice structure. Smaller 
molecules are able to pass through the pores more easily and thus more 
quickly than are larger molecules. Thus, smaller molecules advance a 
greater distance through the gel composition in a given amount of time 
than do larger molecules. The smaller molecules thereby become separated 
from the larger molecules in the process. In this manner DNA fractionation 
occurs. 
While gel electrophoresis is a well known and often used process for DNA 
fractionation, electrophoretic mobility is not well understood in gel 
lattice structures. Thus, the process has several inherent limitations. 
For example, the pore size in the lattice of gels cannot be accurately 
measured or depicted. Therefore, the lengths of the molecules migrating 
through the lattice cannot be accurately measured. It has also been found 
that DNA molecules larger than 20 megabasepairs in length cannot be 
accurately fractionated in gels. Apparently, the pore size in the lattice 
of such materials cannot be increased to permit the fractionation of 
larger molecules, much less even larger particles, viruses, or free cells. 
Further, the lattice structure formed when a gel hardens is not 
predictable. It is not possible to predict the configuration into which 
the lattice structure will form or how the pores therein will be 
positioned, sized, or shaped. The resulting lattice structure is different 
each time the process is carried out. Therefore, controls and the critical 
scientific criteria of repeatability cannot be established. 
Gel electrophoresis experiments cannot be exactly duplicated in order to 
predictably repeat previous data. Even if the exact lattice structures 
formed in one experiment were determinable, the structure could still not 
be reproduced. Each experiment is different, and the scientific method is 
seriously slowed. 
Also, the lattice structure of a gel is limited to whatever the gel will 
naturally produce. The general size of the pores can be dictated to a 
degree by varying the concentration of the gel, but the positioning of the 
pores and the overall lattice structure cannot be determined or designed. 
Distinctive lattice structures tailored to specific purposes cannot be 
created in a gel. 
Further, because the lattice structure arrived at depends upon the 
conditions under which hardening of the gel occurs, the lattice structure 
even in a single composition need not be uniform throughout. 
Another shortcoming of gel electrophoresis is caused by the fact that a gel 
can only be disposed in a layer that is relatively thick compared to the 
pores in its lattice structure, or correspondingly to the size of the DNA 
molecules to be fractionated. Thus, the DNA molecules pass through a gel 
in several superimposed and intertwined layers. Individual DNA molecules 
cannot be observed separately from the entire group. Even the most thinly 
spread gel is too thick to allow an individual DNA molecule moving through 
the gel to be spatially tracked or isolated from the group of DNA 
molecules. 
Once a gel has been used in one experiment, the gel is contaminated and 
cannot be used again. The gel interacts with the materials actually used 
in each experiment, and cleaning of the gel for later reuse is not 
possible. A gel layer must therefore be disposed after only one use. This 
also frustrates the scientific objective of repeatability. 
Finally, simple gel electrophoresis cannot be used to fractionate DNA 
molecules larger than approximately 20 kilobases in length. To overcome 
this fact, it is known to pulse the applied electric field to attempt to 
fractionate longer DNA molecules. This technique, however, results in 
extremely low mobility and requires days of running time to achieve 
significant fractionation. Also, the numerical predictions of the theories 
developed to explain the results of this technique depend critically on 
the poorly known pore size and distribution in the lattice of the gel. 
B. Cell Fractionation 
The flexibility of cells is a structural variable of some interest to cell 
biologists. The flexibility of cells and the effects of various 
environments on cell flexibility is important to the study of the aging 
process in cells. However, cell fractionation based upon cell flexibility 
is not easily accomplished in the prior art. 
For example, various cells have round or oval shapes with various 
diameters. The shapes are often determined by an underlying cytoskeleton. 
When the cells are circulating in the human body, the cells must, on 
several occasions, pass through variously sized openings and passageways. 
This requires substantial flexibility of the cell. The inability to pass 
through these openings can be caused by the aging of a cell, reactions to 
specific chemical environments, and other metabolic changes. When 
referring to red blood cells, poor red blood cell flexibility results in 
serious consequences for the larger organism. With respect to cells such 
as cancer cells, poor flexibility may result in growth and spread of 
tumors. 
Cancer cells are generally thought to settle in the human body in blood 
vessels larger than the cells themselves and stick to those vessels 
through a special adhesion molecule. As the cancer cells stick to the 
vessels, new tumors begin to grow. New information, however, has indicated 
that the cancer cells move too quickly to become adhered to the vessels in 
this fashion. It is now thought that cells may start new tumors when they 
become stuck in vessels too narrow for the cancer cells to pass through. 
The flexibility of the cancer cells is important in determining the 
deleterious effect of the cell. 
Three physical limitations impinge on the flexibility of many cells. First, 
many cells must maintain both a constant volume V and a constant surface 
area A as it deforms. Second, the cell membrane, while very flexible, 
cannot increase in area. It will tear, if forced to do so. Third, as a 
cell ages it loses membrane and the surface-to-volume ratio decreases. 
For example, a biconcave red blood cell has a maximum diameter of about 8 
microns, a surface area of about 140 microns square, and a volume of about 
95 microns cube in the normal state. It can be shown that for mature red 
blood cells for openings smaller in diameter than approximately 3 microns, 
the constraints of constant volume V and surface area A cannot be met. The 
passage of a red blood cell through a passageway of that size, thus, 
cannot occur without membrane rupture. Since the smallest capillary 
openings are but approximately 3.5 microns, red blood cells passing 
through the capillary bed are uncomfortably close to being ruptured. 
Accordingly, small changes in the physical variables that control 
deformability can lead to microangiopathy and severe organism distress. 
There exist several techniques for measuring cell flexibility and 
deformability. These range from the elegant and pioneering micropipette 
aspiration techniques, to the nucleopore filtration and laminar stress 
elongation techniques. The latter are termed ektacytometry. All are very 
useful and have provided an excellent initial database for studying red 
blood cell deformation, but each has certain weaknesses. 
The micropipette aspiration technique can only study one cell at a time. 
The nucleopore filtration technique does not allow observation of cells 
during the actual passage thereof through openings. Ektacytometry does not 
deform cells in narrow passages. 
OBJECTS AND SUMMARY OF THE INVENTION 
It is accordingly a broad objective of the present invention to provide an 
improved method and apparatus for fractionating microstructures, such as 
macromolecules, viruses, free cells, and minute particles. 
Another object of the present invention is to facilitate research into the 
behavior and structure of macromolecules, such as DNA molecules, proteins 
and polymers. 
Correspondingly it is an object of the present invention to enhance the 
effectiveness of electrophoresis techniques currently applied to the 
fractionation of such macromolecules. 
Yet another object of the present invention is to permit fractionation of 
DNA molecules in excess of 20 megabasepairs in length, without resorting 
to the use of a pulsed electric field. 
Yet another object of the present invention is to provide a hindered 
environment in which to conduct macromolecular electrophoresis, wherein 
the lattice structure of the hindered environment can be designed at will 
and replicated with repeatable consistency. 
Another object of the present invention is to provide such a lattice 
structure in which the distribution, size, and shape of the pore therein 
are substantially uniform. 
Yet another object of the present invention is to provide an apparatus for 
fractionating macromolecules while simultaneously observing individual of 
the macromolecules during the process. 
Yet another object of the present invention is to advance the study of the 
structure and mechanics of free cells, such as red blood cells, cancer 
cells, and E. coli cells. 
It is yet another object of the present invention to provide an apparatus 
for fractionating cells according to the elasticity thereof and other 
physical properties which are otherwise difficult to probe by biological 
markers. 
In particular, it is an object of the present invention to provide a method 
and apparatus for observing cell behavior during the passage of cells 
through channels in essentially a single layer in single file. 
Yet another object of the present invention is to provide an apparatus for 
sorting and viewing microstructures, which is not contaminated by the 
microstructures being sorted. 
Yet another object of the present invention is to increase the mobility of 
large molecules during electrophoresis. 
Additional objects and advantages of the invention will be set forth in the 
description which follows, and in part will be obvious from the 
description, or may be learned by the practice of the invention. The 
objects and advantages of the invention may be realized and obtained by 
means of the instruments and combinations particularly pointed out in the 
appended claims. 
To achieve the foregoing objects, and in accordance with the invention as 
embodied and broadly described herein, a sorting apparatus is provided for 
fractionating and simultaneously viewing individual microstructures such 
as free cells, viruses, macromolecules, or minute particles in a fluid 
medium. The sorting apparatus allows the microstructures to be observed in 
essentially a single layer and whereby a particular microstructure can be 
tracked throughout. One embodiment of an apparatus incorporating the 
teachings of the present invention comprises a substrate having a shallow 
receptacle located on a side thereof. The receptacle has first and second 
ends and a floor bounded on opposite sides by a pair of upstanding opposed 
side walls extending between the first and second ends of the receptacle. 
Migration of the microstructures from the first end of the receptacle to 
the second end of the receptacle defines a migration direction for the 
receptacle. The height of the side walls defines a depth of the 
receptacle. The depth is commensurate with the size of the microstructures 
in the fluid medium, whereby the microstructures will migrate in the fluid 
through the receptacle in essentially a single layer. 
According to one aspect of the present invention, the array further 
comprises sifting means positioned within the receptacle intermediate the 
first and second ends traversing the migration direction. The sifting 
means are for interacting with the microstructures to partially hinder 
migration of the microstructures in the migration direction in the fluid 
medium. 
In one embodiment of such a sifting means, an array of obstacles is 
provided upstanding from the floor of the receptacle. The array of 
obstacles is arranged in a predetermined and reproducible pattern. The 
obstacles may comprise posts, bunkers, v-shaped and cup-shaped structures, 
and other shapes of structures. In a preferred embodiment, the receptacle 
and array of obstacles therein are simultaneously formed on a side of the 
substrate using microlithography techniques. 
According to another aspect of the present invention, the apparatus further 
comprises ceiling means positioned over the sifting means for covering the 
receptacle and for causing migration of the microstructures in essentially 
a single layer through the sifting means exclusively. The ceiling means 
are so secured to the sifting means as to preclude migration of 
microstructures between the sifting means and the ceiling means. In one 
embodiment of an apparatus incorporating the teachings of the present 
invention, such a ceiling means comprises a coverslip which extends across 
the substrate from one of the pair of upstanding opposing side walls to 
the other of the pair of upstanding opposed side walls with the tops of 
the obstacles in the array bonded to the adjacent side of the coverslip. 
Optimally, the coverslip and the substrate have similar thermal 
coefficients of expansion. Also, preferably the substrate and the array of 
obstacles are comprised of a material that is noninteractive in a normal 
range of temperatures with the microstructures to be fractionated therein. 
Optionally, the coverslip may be transparent, thereby to afford for visual 
observation of the microstructures during sorting. The transparent form of 
the coverslip represents one example of a structure capable of performing 
the function of what will hereinafter be referred to as a "capping means" 
for the present invention. 
In another aspect of an apparatus incorporating the teachings of the 
present invention, the array includes electric force means for generating 
in the receptacle an electric field used to induce charged microstructures 
to migrate through the fluid medium from one end of the receptacle to the 
other. In one embodiment, such an electric force means may comprise a 
first electrode positioned at the first end of the receptacle and a second 
electrode positioned at the second end of the receptacle. The electrodes 
may comprise metal strips disposed on the floor of the receptacle. A power 
source is electrically coupled between the first and second electrodes. 
In yet another aspect of the present invention, an apparatus incorporating 
the teachings thereof further comprises 
sensor means positioned within the array of obstacles for sensing the 
intensity of the electric field generated within the array. The sensor 
means may optionally be electrically coupled with the electric force means 
to vary the intensity of the electric field in a predetermined manner. In 
one embodiment of the sensor means, first and second sensor electrodes are 
positioned within the array of obstacles, and control means are coupled to 
the first and second electrodes for maintaining the electric field in the 
array at a predetermined intensity. 
In one embodiment of the present invention, such a control means includes a 
differential amplifier circuit having first and second input terminals 
coupled respectively to the first and second sensor electrodes. The 
differential amplifier circuit produces an output signal corresponding to 
the intensity of the electric field in the array between the first and 
second sensor electrodes. Comparator means are coupled to the differential 
amplifier for producing a control signal reflecting the difference between 
the output signal of the differential amplifier and a reference voltage 
reflecting the predetermined intensity of the electric field in the array. 
Driver means are coupled to the comparator means for varying the intensity 
of the electric field in accordance with the control signal produced by 
the comparator means. 
The present invention also contemplates a method for manufacturing an 
apparatus as described above. In the method a receptacle is formed on one 
side of a substrate having a floor bounded by a pair of upstanding 
opposing side walls. An array of obstacles are built within the 
receptacle. Preferably the step of forming the receptacle and the step of 
building the array are performed simultaneously. To do so, a photoresist 
layer is positioned over areas of the substrate intended to correspond to 
the tops of the obstacles of the arrays. Then the substrate is etched to a 
predetermined depth equal to the depth of the receptacle. The receptacle 
with the array of obstacles upstanding therein is formed as a result. The 
photoresist layer is then dissolved from the substrate. 
Ultimately the method of the present invention includes the step of 
securing a transparent coverslip to the top of each of the obstacles. To 
do so the coverslip is positioned over the array of obstacles in contact 
with the top of each. An electric field is applied between the coverslip 
and the array of obstacles. 
The present invention also contemplates a method for sorting and 
simultaneously viewing individual microstructures. In that method the 
microstructures are placed in a fluid medium and introduced into one end 
of an apparatus as described above. The microstructures are then induced 
to migrate in the fluid through the array of obstacles and visually 
observed during the process. 
An additional embodiment within the scope of the present invention 
comprises an apparatus for sorting and simultaneously viewing cells in a 
fluid medium in order to study flexibility of cells and the effects of 
various environment on cells. The apparatus comprises a substrate having a 
shallow receptacle located on a side thereof and channeling means 
positioned within the receptacle for allowing passage of cells through the 
receptacle in essentially a single layer and in single file. In one 
embodiment of the present invention, such a channeling means comprises 
passageways positioned within the receptacle through which the cells may 
pass. 
The apparatus can be used to measure the amount of energy consumed during 
movement of the cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention provides a method and apparatus that facilitates the 
fractionation of many types of microstructures. For example, the present 
invention allows successful fractionation of extremely long DNA molecules 
of chromosomal length in low quantities, such as even single molecules. 
The present invention also facilitates the fractionation of much larger 
microstructures, such as red blood cells. 
Each application will be described in turn below. 
A. Macromolecule Fractionation 
Although reference will be made herein to the fractionation of DNA 
molecules, it should be noted that fractionation of other macromolecules 
and microstructures, such as proteins, polymers, viruses, cells, and 
minute particles, is considered to be within the scope of the present 
invention. 
The diffusion of long polymers in complex environments where the mobility 
of the polymer is greatly perturbed is both a challenging statistical 
physics problem and a problem of great importance in the biological 
sciences. The length fractionation of charged polymers, such as DNA in 
gels, is a primary tool of molecular biology. One of the main stumbling 
blocks to understanding quantitatively the physical principles behind the 
length-dependent mobility of long polymers in complex environments has, 
however, been the ill-characterized nature of the hindering environment, 
the gel. It is possible, however, using the present invention to generate 
complex environments which are very well characterized and consistently 
reproducible. 
Referring to FIG. 1, a sorting apparatus 20 is illustrated for 
fractionating and simultaneously viewing microstructures such as free 
cells, macromolecules, and minute particles in a fluid medium in 
essentially a single layer. Sorting apparatus 20 is comprised of a 
substrate 22 having a shallow receptacle 24 located on a side 26 thereof. 
In the embodiment shown, receptacle 24 is recessed in side 26 of substrate 
22, although other structures for producing a recess such as receptacle 24 
would be workable in the context of the present invention. 
Receptacle 24 includes a floor 28 shown to better advantage in FIG. 2 
bounded by a pair of upstanding opposing side walls 30, 31 and a first end 
32 and a second end 34. The height of side walls 30, 31 define a depth of 
receptacle 24. The depth of receptacle 24 is commensurate with the size of 
the microstructures to be sorted in sorting apparatus 20. The depth of 
receptacle 24 is specifically tailored to cause those microstructures in a 
fluid medium in receptacle 24 to form essentially a single layer. Thus, 
when the microstructures are caused to migrate in the fluid medium through 
receptacle 24, the microstructures do so in essentially the single layer. 
The migration of the microstructures occurs in a migration direction 
indicated by arrow M defined relative to sorting apparatus 20. 
Substrate 22 may be comprised of any type material which can be 
photolithographically processed. Silicon is preferred, however other 
materials, such as quartz and sapphire can also be used. 
In accordance with one aspect of the present invention, ceiling means are 
provided for covering receptacle 24 intermediate first end 32 and second 
end 34 thereof and for causing the migration of the microstructures within 
receptacle 24 to occur in essentially a single layer. As shown by way of 
example and not limitation, in FIG. 1, a coverslip 36 extends across 
receptacle 24 in substrate 22 from one of the pair of upstanding opposing 
side walls 30 to the other of said pair of upstanding opposing side walls 
31. The manner by which coverslip 36 is bonded to side 26 of substrate 22 
will be discussed in detail subsequently. 
According to one aspect of the present invention, a sorting apparatus, such 
as sorting apparatus 20, is provided with sifting means positioned within 
receptacle 24 reversing the migration direction associated therewith for 
interacting with the microstructures to partially hinder the migration of 
the microstructures in the migration direction. 
As is suggested in the exploded view of FIG. 2, one form of such a sifting 
means utilizable in accordance with the present invention is an array 38 
of minute obstacles 39 upstanding from floor 28 of receptacle 24. 
Obstacles 39 are sized and separated as to advance the particular sorting 
objective of sorting apparatus 20. The manner of forming obstacles 39 of 
array 38, as well as a number of examples of embodiments of obstacles 
utilizable in such an array, will be discussed in substantial detail 
below. 
Coverslip 36 is so secured to the top of obstacles 39 in array 38 as to 
preclude migration of microstructures between the obstacles 39 and 
coverslip 36. Coverslip 36 may optionally be transparent. In this form, 
coverslip 36 performs not only the function of the ceiling means described 
above, but also performs the function of a capping means for covering a 
shallow receptacle, such as receptacle 24, and for affording visual 
observation therethrough of the migration of microstructures through array 
38. Coverslip 36 may be comprised of any ceramic material. Pyrex is 
preferred, but other materials such as quartz and sapphire, for example, 
may also be used. 
In accordance with another aspect of the present invention, a sorting 
apparatus, such as sorting apparatus 20, is provided with electric force 
means for generating an electric field in the fluid medium in receptacle 
24. The electric field induces the microstructures to migrate through the 
fluid medium, either from first end 32 to second end 34 or from second end 
34 to first end 32, depending upon the polarity of the electric field and 
whether the microstructures are positively or negatively charged. 
Negatively charged microstructures, such as DNA molecules, will be induced 
to flow toward the positive pole. Positively charged microstructures, such 
as proteins, will be induced to flow toward the negative pole. 
By way of example and not limitation, a first electrode 40 is shown in FIG. 
2 as being located in first end 32 of receptacle 24 and a second electrode 
42 located in second end 34 of receptacle 24. First electrode 40 and 
second electrode 42 each comprise a metal strip disposed on floor 28 of 
receptacle 24. In the preferred embodiment, the metal strip is formed from 
evaporated gold. 
A battery 44, or other power source is electrically coupled between first 
and second electrodes, 40 and 42, such that first electrode 40 comprises a 
negative pole and second electrode 42 comprises a positive pole. The 
electric field generated between first and second electrodes, 40 and 42, 
is non-alternating, but the use of an alternating power source in place of 
battery 44 would be consistent with the teachings of the present 
invention. 
When DNA is the microstructure being induced to migrate, the electric field 
intensity in receptacle 24 is in the range of from about 0.1 volt per 
centimeter to about 10 volts per centimeter. In the preferred embodiment, 
an electric field intensity is about 1.0 volt per centimeter. 
Referring now to FIG. 3, the portion of FIG. 2 encircled by line 3--3 is 
seen illustrated in an enlarged manner. FIG. 3 illustrates one example of 
a sifting means for use in a sorting apparatus of the present invention. 
As shown, array 38 comprises a plurality of obstacles 39 upstanding from 
floor 28 of receptacle 24. Although FIG. 3 illustrates obstacles 39 as 
being positioned within array 38 in an ordered and uniform pattern, it is 
within the scope of the present invention to have a staggered pattern, or 
any desired predetermined and reproducible pattern. 
FIG. 4 illustrates the various dimensions of a typical obstacle 39. The 
height H of obstacle 39 is measured in a direction normal to floor 28 of 
receptacle 24. The length L of obstacle 39 is measured in a direction 
parallel to said migration direction M. The width W of obstacle 39 is 
measured in a direction normal to the migration direction M. Each of the 
obstacles 39 are separated from an adjacent obstacle 39 by a predetermined 
separation distance S.sub.d. The space between adjacent of obstacles 39 in 
a cross section of array 38 taken normal to floor 28 of receptacle 24 
defines a pore 54 of the lattice structure cumulatively produced by 
obstacles 39 of array 38. For convenience of reference in FIG. 4, such a 
typical pore 54 has been shaded, but will be discussed in additional 
detail subsequently. These dimensions can be changed and designed to be as 
desired depending upon the type and size of microstructure to be sorted, 
the design of the array, and the type of obstacles in the array. 
For example, the separation distance S.sub.d will vary depending upon 
whether the migration of microstructures through pores 54 are DNA 
molecules, viruses and bacterial cells, or mammalian cells. For migration 
of DNA molecules, the separation distance S.sub.d is within the range of 
about 0.01 microns to about 20.0 microns. For migration of viruses and 
bacterial cells, the separation distance S.sub.d is within the range of 
about 0.01 microns to about 1.0 micron. For migration of mammalian cells, 
the separation distance is within the range of from about 1.0 micron to 
about 50.0 microns. It is presently preferred that the separation distance 
S.sub.d be substantially equal to the radius of gyration of the molecule, 
the radius of gyration being the distance walking out from the center of 
the molecule. 
Length L also varies depending upon the microstructure to be migrated 
through array 38 of obstacles 39. In a presently preferred embodiment, the 
length is generally equal to the separation distance. With regard to 
height H, the height of obstacles may generally be in the range of from 
0.01 microns to about 20.0 microns. For smaller microstructures, the 
obstacles may have a height in a range from about 0.01 microns to about 
0.50 microns. For larger microns, the height may be in the range from 
about 1.0 micron to about 5.0 microns. 
FIG. 4A, a cross-section of two obstacles 39, illustrates in planar view a 
typical pore 54. Pore 54 compresses the area defined by two obstacles 39 
through which a microstructure must pass. Pore 54 is defined by the height 
H and the separation distance S.sub.d between the obstacles. The desired 
size of pore 54 is determined by reference to the size of the 
microstructures to be sorted therethrough. An important aspect with the 
apparatus of the present invention is that not only is the pore size of 
the arrays known, but it is also constant and reproducible. More stable 
data can be obtained. 
The characteristic number which sets the length scale for the conformation 
of a polymer in solution is the persistence length given by the equation: 
##EQU1## 
E is the Young's modulus, I.sub.A is the surface moment of inertia, 
K.sub.B is Boltmann's constant, and 
T is the absolute temperature. 
For DNA at normal physiological salt concentrations and pH, about 0.1M NaCl 
and pH 7.6, P is 0.06 microns. If the etch depth of the array is 
approximately equal to or less than P then the polymer can be viewed as 
moving in a quasi-two-dimensional environment, as is the case in the 
apparatus used within the scope of the present invention. 
In one preferred embodiment of a sorting apparatus, such as sorting 
apparatus 20, incorporating the teachings of the present invention, 
substrate 22 is provided with a receptacle 24 having sides 30 and 31 of 
approximately 3.0 millimeters in length and first and second ends 32, 34, 
respectively, of approximately 3.0 millimeters in length. Each of 
obstacles 39 has a height H of approximately 0.1 microns, a width W of 
approximately 1.0 micron, a length L of approximately 1.0 micron and a 
separation distance S.sub.d of approximately 2.0 microns. These sizes will 
vary depending upon the microstructure to be sorted, bearing in mind that 
obstacles 39 should be so sized and separated in array 38 that 
microstructures migrate through array 38 of obstacles 39 in essentially a 
single layer. 
The method of making the apparatus of the present invention involves 
forming receptacle 24 on one side of substrate 22. Receptacle 24 should be 
formed of a size such that microstructures migrate in the fluid through 
receptacle 24 in essentially a single layer. A further step comprises 
creating array 38 of obstacles 39 within receptacle 24. Each of obstacles 
39 have a top 56, sides 57, and a bottom end 58. Obstacles 39 are 
upstanding from floor 28 of receptacle 24 in a predetermined and 
reproducible pattern. In one preferred embodiment, the array of obstacles 
comprises a plurality of posts. 
By way of example and not limitation, the creation of posts within the 
receptacle is illustrated in FIGS. 5A-5F. As shown in FIG. 5A, the forming 
step comprises developing a photosensitive photoresist layer 60 over areas 
of substrate 22 that are intended to correspond to tops 56 of obstacles 
39. This is accomplished by exposing substrate 22 to light through a mask 
having thereon a corresponding opaque pattern. 
The portion of photoresist layer 60 which is exposed to light becomes 
soluble in a basic developing solution, while the unexposed portion 
remains on substrate 22 to protect substrate 22. Thus, after development 
in the developing solution, substrate 22 is left with a pattern of 
photoresist layer 60 that is identical to the opaque pattern of the mask. 
FIG. 5B illustrates substrate 22 with photoresist layer 60 thereon after 
exposure to light and development in solution. 
The next step comprises etching substrate 22 such that the areas of 
substrate 22 unshielded by photoresist layer 60 are exposed to the 
etching, thereby forming receptacle 24. The array 38 of obstacles 39 
upstanding within the etched receptacle 24 is formed by the portions of 
substrate 22 shielded by photoresist layer 60. FIG. 5C illustrates 
formation of receptacle 24 and the obstacles 39. 
As can be seen in FIG. 5C, as the substrate 22 is etched, the photoresist 
layer 60 is also etched, but at a slower rate. FIG. 5C illustrates the 
receptacle 24 half formed, and photoresist layer 60 partially etched away. 
If, for example, the photoresist layer is etched at a rate 1/10 the rate 
that substrate 22 is etched, the resulting receptacle can at most have a 
depth 10 times the thickness of the photoresist layer. The thickness of 
photoresist layer 60 must therefore be chosen accordingly. 
The etching process can be terminated at any time when the desired depth of 
the receptacle is reached. As illustrated in FIG. 5D, there may be some 
photoresist layer 60 still present on substrate 22 when the etching is 
terminated. If so, the next step is then dissolving photoresist layer 60 
from substrate 22. This step leaves a clean substrate 22 as shown in FIG. 
5E. 
Within the scope of the present invention, etching may occur by many types 
of methods. In the preferred embodiment, ion milling is used such that an 
overhead ion beam is used to etch the substrate 22 and photoresist layer 
60. Other methods of etching, such as chemical etching, are also within 
the scope of the present invention. 
Turning now to FIG. 5F, the step of fusing coverslip 36 to substrate 22 is 
illustrated. In the preferred embodiment within the scope of the present 
invention, the step comprises positioning coverslip 36 over array 38 of 
obstacles 39 such that coverslip 36 is in contact with each of obstacles 
39, and then applying an electric field between coverslip 36 and each of 
obstacles 39. The coverslip 36 is held with a negative potential. The 
obstacles 39 are held at a positive potential. Ions are thereby induced to 
migrate there between to create a bond between coverslip 36 and each of 
obstacles 39 at all areas of contact. The process of this step is referred 
to as field assisted fusion. 
The voltage used to fuse coverslip 36 to the substrate 22 is preferably 
about 1 kilovolt but can be within the range of from 200 volts to about 
2000 volts. The time for fusion is about 30 minutes at a temperature of 
about 400.degree. C. The temperature can also range from about 300.degree. 
C. to about 600.degree. C., with 400.degree. C. being the preferred 
temperature. In the preferred embodiment within the scope of the present 
invention, the coverslip comprises a pyrex material. However, any 
transparent ceramic may be used. For example, sapphire and quartz are 
material which may also be used for the coverslip. 
It is preferred that the material used for coverslip 36 have substantially 
the same coefficient of thermal expansion as substrate 22. Otherwise, at 
the high temperature of fusion, the coverslip 36 and the substrate 22 will 
expand at different rates and a seal between the two would be difficult or 
impossible to accomplish. 
Successful fusion can be tested by injecting a fluorescent fluid into the 
apparatus. A completely fused coverslip will not allow passage of any 
fluorescent fluid between coverslip 36 and obstacles 39. 
FIG. 6 illustrates one use of an embodiment of the present invention. As 
earlier stated, the apparatus of the present invention can be used for 
charged macromolecular electrophoresis. For example, the apparatus may be 
used to conduct protein electrophoresis, and DNA electrophoresis, with the 
positive and negative poles adjusted accordingly. FIG. 6 illustrates DNA 
electrophoresis. 
As illustrated in FIG. 6 by way of example and not limitation, DNA 
molecules 68 are placed into a buffer solution and placed into a loading 
area 66 positioned on the first end 32 of receptacle 24. Loading area 66 
comprises a portion of receptacle 24 where no obstacles 39 have been 
formed. Buffer is also added to a second loading area 67 positioned on 
second end 34. Second loading area 67 also comprises a portion of 
receptacle 24 where no obstacles have been formed. The loading areas are 
then covered. 
Once DNA molecules 68 have been positioned, battery 44 is engaged and an 
electric field is generated. The electric field is so polarized as to 
induce the negatively charged DNA microstructures to migrate through the 
field from first electrode 40 toward second electrode 42 in receptacle 24. 
As DNA molecules 68 migrate from first end 32 toward the second end 34, 
their movements are hindered by the array 38 of obstacles 39 upstanding 
within receptacle 24. Interaction between obstacles 39 and DNA molecules 
68 are illustrated in FIG. 6. 
In FIG. 6, DNA molecules 68 are illustrated as long arrows. The direction 
of the arrows indicates the direction of migration of DNA molecules 68. As 
DNA molecules migrate through array 38 of obstacles 39, large bodies of 
DNA molecules may become hooked by obstacles 39 and may become trapped. 
The hooked and trapped DNA molecules are labelled as 68a. When, as 
illustrated in FIG. 6, obstacles 39 are posts, DNA molecules 68 stretch 
around obstacles 39 as they become hooked. The obstacles are thought to 
catch the large DNA molecules and hold them against the electric field. 
Some DNA molecules 68 may stretch and release themselves from the 
obstacles. Smaller DNA molecules possess sufficient Brownian motion to 
release themselves. 
It is an important feature of the present invention that any pattern of 
array 38 of obstacles 39 can be designed within the scope of the present 
invention. The array 38 can comprise an ordered, evenly spaced formation 
wherein the obstacles are positioned in uniform rows and columns. 
Alternatively, array 38 may comprise a staggered formation wherein 
positioning of the obstacles is not uniform but rather scattered around 
the array. Further, array 38 may comprise a mixture of such arrangements 
disposed along migration direction M traversing same. 
The design of the array can be formulated to correspond to any specific 
intended use. The ordered, evenly spaced configuration can be used for 
imaging of long megabase DNA fragments. The staggered configuration, 
having a higher possibility of hooking the DNA molecules as the DNA 
molecules migrate through the array, can be used to more directly test the 
role of DNA relaxation and hooking in the mobility of DNA molecules. 
The shapes of the obstacles may also vary within the scope of the present 
invention. Illustrated in FIG. 7 is an array 70 of v-shaped obstacles 72 
upstanding from floor 28 of receptacle 24, and having a v-shaped cross 
section in a plane disposed parallel to floor 28 of receptacle 24. Arms 73 
and 74 intersect at one end to form a vertex 75 and an open end 76. The 
open end 76 of said v-shaped cross section of v-shaped obstacles 72 is 
disposed opposing migration direction M of receptacle 24. 
The size of v-shaped obstacles 72 should be such that as microstructures of 
various sizes migrate through the array 70 of v-shaped obstacles 72 in a 
direction M, the microstructures are hindered and trapped within the open 
end 76 of v-shaped obstacles 72. Smaller v-shaped obstacles 72 will trap 
small microstructures while larger v-shaped obstacles 72 will trap both 
the smaller and the larger microstructures. 
It is conceivable that various sizes of v-shaped obstacles 72 may be used 
within one array 70. For example, smaller v-shaped obstacles 72 may be 
positioned toward the first end 32 of receptacle 24 with larger v-shaped 
obstacles 72 positioned toward the second end 34 of receptacle 24. Thus, 
as the microstructures migrate from first end 32 toward second end 34, the 
smaller microstructures will become trapped in the smaller v-shaped 
obstacles 72 while the larger microstructures will flow past the smaller 
v-shaped obstacles 72. As the larger microstructures flow through the 
larger sized v-shaped obstacles 72, the larger microstructures will also 
become trapped. The microstructures will then be separated with respect to 
size. 
Referring now to FIG. 8, an alternate embodiment of the array of obstacles 
within the scope of the present invention is illustrated. FIG. 8 
illustrates an array 78 of obstacles 80 which are cup-shaped. Obstacles 80 
have a cup-shaped cross section in a plane disposed parallel to floor 28 
of receptacle 24. 
As illustrated, cup-shaped obstacles 80 may comprise a first leg 82 and a 
second leg 84 substantially parallel to the direction of migration of the 
microstructures, and a third leg 86 substantially perpendicular to the 
direction of migration. First, second, and third legs, 82, 84, and 86, 
respectively, are positioned such that they define an open end 88 into 
which the microstructures can become trapped as the microstructures 
migrate through the cup-shaped obstacles 80. As with v-shaped obstacles 
72, various sizes of cup-shaped obstacles 80 may be positioned within 
array 78 in any pattern desired. The open end 88 of the cup-shaped 
cross-section is disposed opposing migration direction M of receptacle 24. 
It is important to note that whatever type of array is used, the array is 
reproducible. Additionally, an optimum design can be perfected over time 
by making minor changes to the arrays for each new experiment until the 
most preferred design is obtained. 
Referring now to FIG. 9, and in accordance with another aspect of the 
present invention, a sorting apparatus 110 is comprised of an apparatus, 
such as sorting apparatus 20, further provided with sensor means for 
detecting the intensity of the electric field generated within the array 
of obstacles, such as array 38 of obstacles 39, between any determined 
first and second points therein, to enable control of the intensity of the 
electric field. 
Sorting apparatus 110 is illustrated in FIG. 9. As in sorting apparatus 20, 
shown in FIGS. 1 and 2, sorting apparatus 110 includes first electrode 40 
and second electrode 42, functioning as negative and positive poles, for 
an electric field generated therebetween. That field may be 
non-alternating, by coupling therebetween a battery, such as battery 44 of 
FIGS. 1 and 2. Nevertheless, it would also be consistent with the 
teachings of the present invention to develop an electric field that is 
alternating or switchable as to polarity, either selectively or according 
to some repeated pattern. In the case of sorting apparatus 110, however, 
the electric field developed between first and second electrodes 40 and 42 
is produced by a feedback varied drive voltage circuit 144 that will be 
explored in detail subsequently. 
First electrode 40 comprises a metal strip positioned along floor 28 of 
receptacle 24 at first end 32. First electrode 40 is soldered to substrate 
22 and to various lead lines at a first area 128. Second electrode 42 
comprises a metal strip positioned along floor 28 of receptacle 24 at 
second end 34. Second electrode 42 is soldered to substrate 22 and to 
various lead lines at a second area 129. In the preferred embodiment, the 
metal strips, first and second electrodes 40 and 42, comprise gold 
evaporated into floor 28. 
Positioned within the array is sensor means for detecting the intensity of 
the electric field generated between first electrode 40 and second 
electrode 42 between predetermined first and second points therein. The 
sensor means enables control of the intensity of the electric field 
generated. 
The sensor means comprises a first sensor electrode 130 positioned within 
array 38 of obstacles 39 at the first predetermined point 134. The sensor 
means further comprises a second sensor electrode 132 which is positioned 
within array 38 of obstacles 39 at the second predetermined point 135. 
First sensor electrode 130 is positioned within array 38 toward first end 
32 of receptacle 24 in a first sensor channel 138 formed along floor 28 of 
receptacle 24. No obstacles 39 are present within channel 138. A clear 
area is formed wherein the sensor electrode is positioned. 
In one embodiment of the present invention, the array 38 is turned at a 45 
degree angle before the sensor electrodes are positioned within the array. 
As can be seen in FIG. 9, first and second sensor electrodes, 130 and 132, 
extend through sidewall 31 of receptacle 24, past coverslip 36, and onto 
substrate 22. Positioning of first sensor electrode 130 can be seen in 
FIGS. 10-12. 
In FIG. 10, first sensor electrode 130 is shown disposed along floor 28 of 
receptacle 24 within first sensor channel 138. Obstacles 39 can be seen 
positioned along the sides of top sensor channel 138, but not within 
channel 138 itself. Coverslip 36 is shown fused to the obstacles 39 and 
covering channel 138. 
FIG. 11 illustrates channel 137 extending away from sidewall 31 of 
receptacle 24. Obstacles are not present within channel 137. Coverslip 36 
is illustrated in FIG. 11 as positioned over channel 137. 
FIG. 12 illustrates the first sensor soldering area 140 where first sensor 
electrode 130 is soldered to the substrate 22 and connected to first 
sensor lead 152, to be later discussed in more detail. 
Although cross sections for only first sensor electrode 130 are shown, it 
must be noted that second sensor electrode 132 is positioned within 
apparatus 110 in the same fashion. Second sensor electrode 132 is 
positioned within a bottom sensor channel 139 within the array 38 of 
obstacles 39. Second sensor electrode 132 is soldered to substrate 22 and 
connected to a second sensor lead 154 at a second sensor soldering area 
142. Second sensor lead 154 will be later discussed in more detail. 
First electrode 40 is electrically coupled to drive voltage circuit 144 by 
first electrode lead 146 soldered to first electrode 40 at a first 
electrode soldering area 128. Second electrode 42 is grounded by way of a 
first ground lead 148 that is connected to second electrode 42 at a second 
electrode soldering area 129. 
First and second sensor electrodes, 130 and 132, are electrically coupled 
to each other and to drive voltage circuit 144 through a feedback circuit 
150. A first sensor electrode lead 152 connects the first sensor electrode 
130 to feedback circuit 150. A second sensor electrode lead 154 connects 
the second sensor electrode 132 to feedback circuit 150. 
A second ground lead 156 connects feedback circuit 150 to the ground. A 
control lead 158 connects feedback circuit 150 to drive voltage circuit 
144. 
As shown by way of example, the specific structural details of one 
embodiment of a feedback circuit, such as feedback circuit 150 in FIG. 9, 
and a drive voltage circuit, such as drive voltage circuit 144 in FIG. 9, 
can be appreciated by reference to FIG. 13. 
As shown in FIG. 13 for purposes of illustration, receptacle 24 is filled 
with a liquid medium in which the input voltage V.sub.I supplied between 
first electrode 40 and grounded second electrode 42 creates an electric 
field. 
The actual voltage V.sub.A created in the liquid medium in receptacle 24 
between first sensor electrode 130 and second sensor electrode 132 is 
illustrated as a voltage drop occurring over a variable resistor 159. 
Resistor 159 represents the resistance to the electric field presented in 
the liquid medium in receptacle 24 between the first and second 
predetermined points in array 38. In operation of a sorting apparatus such 
as sorting apparatus 110, the composition of the liquid medium will vary 
from a number of causes. This as a result varies the electrical resistance 
of the liquid medium. 
The actual voltage V.sub.A inherently differs from the input voltage 
V.sub.I by the amount of voltage drop occurring in the liquid medium at 
two locations. These are between first electrode 40 and first sensor 
electrode 130 and between second sensor electrode 132 and second electrode 
42. The resistance in the liquid medium in receptacle 24 between first 
electrode 40 and first sensor electrode 130 is illustrated as a resistor 
160a, while the corresponding resistance between second sensor electrode 
132 and second electrode 42 is illustrated as a resistor 160b. 
FIG. 13 illustrates in addition an exemplary arrangement of circuit 
elements intended to perform the functions of drive voltage circuit 144 
and feedback circuit 150 illustrated in FIG. 9. 
In an aspect of the present invention discussed relative to sorting 
apparatus 20, a sorting apparatus, such as sorting apparatus 110, is also 
provided with electric force means for generating the electric field in 
the fluid medium in receptacle 24. In sorting apparatus 20 illustrated in 
FIG. 1, one example of such an electric force means was illustrated in the 
form of battery 44. 
In FIG. 9, however, an alternative form of such an electric force means is 
illustrated in the form of drive voltage circuit 144. Shown in more detail 
in FIG. 13, drive voltage circuit 144 comprises an original voltage 
V.sub.0 which is coupled through an input resister 161 to the negative 
terminal of a differential amplifier 162. In this manner, the voltage 
appearing on first electrode lead 146 coupled to the output terminal of 
differential amplifier 162 has an inverse polarity relative to input 
voltage V.sub.0. A biasing resister 163 is coupled in parallel between the 
negative input terminal of differential amplifier 162 and the output 
terminal thereof. 
While in some embodiments, input voltage V.sub.0 may comprise a battery, it 
is also the intention in sorting apparatus 110 to afford for an input 
voltage V.sub.0, which can itself be variable and which, due to the 
coupling thereof through the negative input terminal of differential 
amplifier 162, is inversely variable relative to the input voltage V.sub.I 
that is eventually supplied over first electrode lead 146 to first 
electrode 40. 
According to one aspect of the present invention, a sorting apparatus, such 
as sorting apparatus 110 illustrated in FIG. 9, includes sensor means for 
detecting the intensity of the electric field generated within the liquid 
medium in receptacle 24 in any preselected portion of array 38. The 
electric field detected corresponds to actual voltage V.sub.A illustrated 
in FIG. 13. In FIG. 13 the preselected portion of array 38 over which 
actual voltage V.sub.A is measured is located between a first 
predetermined point 134 in array 38 corresponding to first sensor 
electrode 130 and a second predetermined point 135 therein corresponding 
to second sensor electrode 132. 
FIG. 13 illustrates an example of circuit elements capable of performing 
the function of such a sensor means for use in a sorting apparatus 
incorporating teachings of the present invention. These elements include 
first sensor electrode 130 positioned within array 38 of obstacles 39 at 
first predetermined point 134 and a second sensor electrode 132 positioned 
within array 38 at second predetermined point 135. In combination 
therewith, the sensor apparatus according to the teachings of the present 
invention comprises control means coupled to first sensor electrode 130 
and second sensor electrode 132 for maintaining the electric field in the 
liquid medium in receptacle 24 at a predetermined intensity. 
The elements of one embodiment of such a control means are shown in FIG. 13 
in the form of the circuit components and functional groupings thereof 
that comprise feedback circuit 150. Feedback circuit 150 functions to vary 
the voltage supplied by drive voltage circuit 144 to first electrode 40 
utilizing a control signal supplied thereto over control lead 158. While 
the elements of feedback circuit 150 will be described in detail 
subsequently, the effect of the control signal supplied over control lead 
158 to drive voltage circuit 144 will be better appreciated fully by an 
initial discussion of the constituent elements of drive voltage circuit 
144. 
The control signal from control lead 158 is applied to the positive input 
terminal of differential amplifier 162 through a second input resistor 
164. The effect of the control signal on control lead 158 is to vary the 
output of drive voltage circuit 144 on first electrical lead 146 with the 
object of stabilizing actual voltage V.sub.A. To do so the intensity of 
the electric field in the fluid medium in receptacle 24 is increased, when 
the control signal indicates that the actual voltage V.sub.A is less than 
some predetermined referenced voltage desired by the operator of sorting 
apparatus 110. Correspondingly, the control signal of control lead 158 is 
oppositely polarized and thus decreases the intensity of the electric 
field in the liquid medium in receptacle 24, when the control signal 
reflects that the actual voltage V.sub.A is greater than that same 
predetermined reference voltage. In this manner, the control signal 
supplied on control lead 158 to drive voltage circuit 144 will by the 
action of differential amplifier 162 adjust the actual effect of original 
voltage V.sub.0 so as to maintain the actual voltage V.sub.A at any 
desired level. 
The use of the control signal supplied over control lead 158 to drive 
voltage circuit 144 could be utilized as a mechanism for effecting desired 
variations in the voltage supplied to first electrode 40 on first electric 
lead 146. Under most circumstances, however, it is anticipated that the 
known propensity of a liquid medium in which microstructures are migrating 
will vary during the time of operation due to a number of factors, such as 
evaporation, chemical reactions, and temperature changes. An initial 
objective of the circuitry that will now be described relative to feedback 
circuit 150 is to compensate for what is in effect the changeable nature 
of the liquid medium in receptacle 24 as illustrated by variable resistor 
159. In this manner actual voltage V.sub.A is maintained at some 
predetermined constant intensity. 
As illustrated in FIG. 13, feedback circuit 150 includes a differential 
amplifier circuit 166 having a first input terminal 167, a second input 
terminal 168, and an output terminal 169. First input terminal 167 is 
coupled through a first buffer amplifier circuit 170 to first sensor 
electrode 130, while second input terminal 168 is coupled through a second 
buffer amplifier circuit 171 to second sensor electrode 132. 
First buffer amplifier circuit 170 is comprised of a differential amplifier 
172 connected in the manner illustrated between the circuit components 
already described above. Correspondingly, second buffer amplifier circuit 
170 is comprised of a differential amplifier 173 connected as illustrated. 
It is the function of first and second amplifier circuits 170, 171, 
respectively, to serve as impedance buffers for first and second input 
terminals 167, 168, respectively, of differential amplifier circuit 166. 
Within differential amplifier circuit 166, first input terminal 167 is 
coupled through an input resistor 174 to the negative input terminal of a 
differential amplifier 175, while second input terminal 168 is coupled to 
the positive terminal thereof through an input resistor 176. Resistors 177 
and 178 are connected as shown in FIG. 13 to bias differential amplifier 
175 into the desired operator thereof. By the arrangements illustrated and 
described, differential amplifier circuit 166 produces at output terminal 
169 thereof an output signal that corresponds to the intensity of actual 
voltage V.sub.A of the electric field in the liquid medium in receptacle 
24. 
According to another aspect of the present invention, a feedback circuit, 
such as feedback circuit 150, includes a comparator means coupled to 
output terminal 169 of differential amplifier circuit 166 for producing a 
control signal at control lead 158 that reflects the difference between 
the output signal on output terminal 169 and a reference voltage 
reflecting a predetermined desired intensity of actual voltage V.sub.A. 
As shown by way of example and in FIG. 13, such a reference voltage is 
supplied by a reference voltage circuit 179 which comprises a differential 
amplifier 180 having a reference voltage V.sub.R coupled to the positive 
input terminal thereof through a variable resistor 181. In this manner, 
variable resistor 181 can be used to adjust the effect of reference 
voltage V.sub.R appearing at the output side of differential amplifier 180 
at an output terminal 182 for reference voltage circuit 179. 
It is the purpose of comparison circuit 183 illustrated in FIG. 13 to 
produce on control lead 158 a control signal reflecting the difference, if 
any, between the output signal appearing at output terminal 169 of 
differential amplifier circuit 166 and the portion of reference voltage 
V.sub.R appearing at output terminal 182 of reference voltage circuit 179. 
Toward that end, comparison circuit 183 comprises a differential amplifier 
184 coupled at the output terminal thereof to control lead 158. The 
positive input terminal of differential amplifier 184 is coupled through 
an input resistor 185 to output terminal 169 of differential amplifier 
circuit 166, while the negative input terminal of differential amplifier 
184 is coupled through an input resistor 186 to output terminal 182 of 
reference voltage circuit 179. Variable resistors 187, 188 are connected 
as shown within comparison circuit 183 to effect desired biasing of 
differential amplifier 184. 
In the circuitry illustrated in FIG. 13, differential amplifiers 162, 172, 
173, 175, 180, and 184 can, by way of example, comprise operational 
amplifiers available from Analog Devices as Product No. AD795N. Such 
devices utilize field effect transistor inputs and have low noise 
characteristics. The values of the resistors illustrated are as follows: 
R.sub.1 =10 k.OMEGA. 
R.sub.2 =10.sup.6 .OMEGA. 
For an apparatus, such as sorting apparatus 110, original voltage V.sub.0 
is equal to negative 15 volts, while reference voltage V.sub.R is equal to 
positive 15 volts. 
By means of the circuitry illustrated in FIG. 14, any desired predetermined 
actual voltage V.sub.A can be maintained between first and second sensor 
electrodes 130, 132, respectively, despite variations over time in the 
nature of the liquid medium in receptacle 24. 
It must be noted that although an electric field has been described in 
detail as the means for inducing migration of the microstructures, other 
fields such as hydrodynamic, magnetic, and gravity, for example, may also 
be used. 
B. Cell Fractionation 
FIGS. 14-18 illustrate another use of the teachings of the present 
invention to facilitate the study of the motion of cells, such as human 
red blood cells, bacterial cells, and cancer cells, for example, through 
channels in a single layer and in single file. For red blood cells, the 
channels may simulate those found in capillaries, the lung alveoli, and 
the spleen in the human body. Further, with the apparatus of the present 
invention, red blood cells can be fractionated on the basis of physical 
properties which are otherwise difficult to probe by biological markers. 
The apparatus within the scope of the present invention comprises 
channeling means positioned within receptacle 24 for allowing passage of 
cells through receptacle 24 in essentially a single layer and in single 
file. 
One possible configuration of an array for all fractionation within the 
scope of the present invention is illustrated in FIG. 14. This array 192 
is called a percolating array and is patterned as a maze. In this 
configuration, the channeling means comprises obstacles 193 positioned 
upstanding from floor 28 of receptacle 24 in various connecting positions 
to form open areas 194, passageways 196, and dead ends 197, such as are 
found in mazes. As can be seen in FIG. 14, cells 199 migrate through 
percolating array 192 through open areas 194 and passageways 196 and are 
at times blocked by dead ends 197. Passageways 196 may be made linear, 
curved, or whatever shape desired so as to be able to observe migration of 
cells through variously shaped passageways. Passageways 196 may have a 
width in the range of from about 1.0 micron to about 10.0 microns and a 
depth with the range of from about 1.0 micron to about 10.0 microns. Cells 
migrating in single file can be seen labelled as 199a. 
Percolation, as herein discussed, is the phenomenon of increasing path 
connectedness due to random addition of discrete segments to allowed 
motion. At the percolation threshold, there is just one path on the 
average through the array, with all other paths leading to dead ends. The 
ability of cells to find that path can be observed with the percolating 
arrays 192 of the present invention. 
Within the scope of the present invention, percolating arrays 192 have been 
constructed on a rectangular lattice in a preferred percolating 
embodiment. A single computer algorithm fills some fraction of the lattice 
with lines, for example, 40% so as to form the variety of open areas 194 
and passageways 196. The computer program is then made into the opaque 
mask and the microlithographic process as earlier described is carried 
out. 
In the preferred embodiment, the obstacles 193 are comprised of barriers 
5.0 microns long and 1.0 micron wide. The preferred etch depth of 
percolating array 192 is 0.35 microns. FIG. 14 illustrates an enlarged 
section of such a photomicrograph percolating array 192. 
One example of the use of percolating array 192 is for study of the 
movements of cells, such as E. coli, from one end of array 192 to the 
other. In one experiment, E. coli cells were placed at the first end 32 of 
receptacle 24 while food was placed at the second end 34 of receptacle 24. 
The E. coli cells were then observed as they migrated in a single layer 
through percolating array 192 from first end 32 toward second end 34. When 
dead ends 197 were reached by the E. coli cells, the manner in which the 
E. coli cells reoriented themselves in order to move away from the dead 
ends 197 was observed. Also observed was the ability of the E. coli cells 
to find an open path from the starting point at first end 32 to the food 
at second end 32. 
The studies conducted for the E. coli cells can also be conducted for many 
other types of cells. Percolating arrays 192 can be used to study the 
manner in which many other types of free floating cells reorient 
themselves in a fluid suspension when confronted with barriers and 
passageways, and the manner in which various passageways are chosen. 
As earlier stated, the percolating arrays 192 are formed such that 
migration of a cell in a single layer and single file can be observed. 
Therefore, in order to accommodate the various sizes of cells to be 
observed, the size of open areas 194 and passageways 196 in each array 192 
can be designed as needed. The pattern of the array can also be designed 
as desired. Any pattern can be produced and reproduced. 
One additional important aspect of percolating arrays 192 is the ability to 
perform electrophoresis of charged spherical balls within percolating 
arrays 92. The mobilities of even simple balls are rich in a percolating 
array because of the numerous dead-ends that exist in a percolating array 
near the percolating threshold. If the electric fields are too big, then 
the balls cannot back-diffuse out of the dead end against the applied 
electric force. Hence, mobility shuts down above a critical field. 
Measuring the diffusion of fluorescent balls of precise diameter will 
allow study of a diffusion of polymers in arrays. 
Referring now to FIG. 15, another embodiment of the present invention can 
be seen. In FIG. 15, an array 200 of obstacles in the form of elongated 
rectangular bunkers 202 is positioned within receptacle 24. Bunkers 202 
are comprised of a rectangular shape having opposing sidewalls 203 and a 
top 204. Bunkers 202 upstand from floor 28 of receptacle 24. Bunkers 202 
are positioned within columns and rows within receptacle 24. Cells migrate 
through the columns and between the rows of bunkers 202 in a migration 
direction indicated by arrow M. The longitudinal axis of the bunkers are 
disposed in alignment with migration direction M. Channels 206 are formed 
between rows of bunkers 202 through which the cells migrate. A separation 
distance, S.sub.r, between rows of bunkers 202, indicates the size of 
channels 206. 
While the size and organization of bunkers 202 may vary, in a preferred 
embodiment within the scope of the present invention, the separation 
distance S.sub.r is sized to allow the cells to migrate through channels 
206 in essentially a single layer in single file. 
The height H of each bunker 202 should also be such that it allows the 
cells to pass through the bunkers 202 in essentially a single layer. As 
with the apparatus for fractionating DNA, a coverslip 36 is fused to the 
tops 204 of bunkers 202 so as to prevent migration of cells between the 
coverslip and the tops 204 of bunkers 202 to ensure that the cells migrate 
through the array 200 of bunkers 202 in essentially a single layer. 
While bunkers 202 are the preferred obstacles for forming channels 206, 
different structures may also be used to simulate channels through which 
the cells can migrate and be observed. These alternate structures are also 
within the scope of the present invention. 
FIG. 16 illustrates an apparatus 212 for cell sorting and fractionation. As 
an example, and not as a limitation, cells 214 are shown migrating through 
array 200 of bunkers 202. Cells 214 can be seen moving between the rows of 
bunkers 202 through channels 206. Some cells begin round, deform to fit 
within channels 206, and then regain their shape once out of the channel. 
Other cells which may have lost some degree of deformability, however, do 
not regain their shape, or are misshapen initially. Some are even trapped 
in these, restricted channels. This, as earlier stated, can be caused by 
aging, sickling or other in vivo or in vitro problems. 
For illustration, cells 214 are shown to be disc shaped. As cells 214 enter 
channels 206, cells 214 deform from a disc shape to an elongated shape so 
as to be able to squeeze through channels 206. When cells 214 are 
positioned between bunkers 202 and within channels 206, cells 214 have a 
thin elongated shape. As cells 214 move from between bunkers 202 and into 
open space, the healthy cells 214 can be seen to resume their original 
disc shapes. The unhealthy cells may be found to not be able to resume 
their original shapes because of a lack of plastic flow. By the apparatus 
of the present invention, the flexibility and deformability of red blood 
cells can be studied. 
FIGS. 17A-17E illustrates an individual cell 214 moving through a pair of 
bunkers 202. As shown in FIG. 17A, before passing through bunkers 202, the 
cell 214 is perfectly disc shaped. In FIG. 17B, cell 214 is seen beginning 
to deform in order to fit between bunkers 202 in channel 206. FIG. 17C 
illustrates cell 214 deformed into an elongated thin shape to fit within 
channel 206. As shown in FIG. 17D, as cell 214 begins to move out of 
channel 206, cell 214 begins to regain its original disc shape. Once 
completely out of channel 206, as shown in FIG. 17E, the elasticity of 
cell 214 allows cell 214 to completely regain its original disc shape. 
In contrast, FIGS. 18A-18B illustrate an unhealthy cell 216 whose elastic 
properties have been lost. Although unhealthy cell 216 has an original 
round disc shape of a healthy cell, its flexibility is diminished such 
that it cannot deform to fit into channel 206. As cell 216 passes through 
channel 206, cell 216 cannot deform into a thin elongated shape to fit 
into channel 206 and becomes stuck in the opening of channel 206. In the 
case of cancer cells, it is thought that where the cancer cells become 
stuck, a new tumor is grown. The activity of cancer cells can be studied 
with the teachings of the present invention. 
Thus, it can be seen that by using the apparatus of the present invention, 
the elasticity and flexibility of cells can be studied. Further, the 
consequences of lack of plastic flow of the cells can be observed and 
studied. Further, still, the amount of energy consumed by the cell to 
deform and regain its shape can easily be measured and recorded. 
Another important advantage and use of the present invention is to study 
and observe the physical properties of cells in a variety of chemical 
environments. Array 200 can be exposed to various chemical environments, 
such as irradiation, light illumination, or sickling phenomena imitations, 
before allowing the cells to migrate through array 200. The reactions of 
cells as they migrate through these various environments can then be 
studied. For example, experiments can be designed to determine what kinds 
of chemical reactions cause aging of the cells and destroy ability of 
cells to be flexible. Other experiments can be designed and conducted to 
determine the chemical effects on cancer cells. Ultimately, an unlimited 
number of cellular effects can be observed. 
Also advantageous, the experiments can be easily repeated to verify data or 
to make minor changes to the experimental controls. Thus, cells can be 
sorted by desired physical properties, that is, by their reactions to 
various environments. As the cells are sorted, they can be separated and 
collected. 
Another important advantage of the present invention with regard to 
studying cells is the reproducibility and repeatability of the array of 
obstacles. Since the arrays 200 can consist of obstacles which are 
repeated thousands of times, even subtle variations in small quantities in 
the membrane of the cell can be amplified. Additionally, by the apparatus 
of the present invention, many individual cells can be observed at once as 
they migrate through the channels 206 of the apparatus. Observation of 
more than just one cell is possible. 
With regard to mobility of the cells through the apparatus of the present 
invention, cells can be migrated through array 200 using various fields. 
For example, migration can be caused by flowing fluid through the array in 
a hydrodynamic field through flow cytometry wherein water pressure is used 
to force the cells through the array. The cells may also be induced to 
move by a gravity field. Alternatively, magnetic beads may be placed on 
the apparatus to create a magnetic field to induce movement of the cells. 
Further, focused beams of light referred to as optical tweezers may be 
used to move the cells through array 200. Other means for inducing the 
cells to migrate through the array 200 are also within the scope of the 
present invention. 
As one example of an embodiment within the scope of the present invention, 
the apparatus can be designed to simulate capillaries in the human body by 
having channeling means positioned within receptacle 24 which mimic the 
openings that the blood cell must pass through in the body. By precise 
control of chemical environment, channel opening and topology, flow 
velocity, and the application of theories of membrane physics, 
understanding can be obtained of how cells pass through complex 
environments, cell aging, and how the chemical environment of the cell 
solution controls the membrane properties. 
The invention may be embodied in other specific forms without departing 
from its spirit other essential characteristics. The described embodiments 
are to be considered in all respects only as illustrative and not 
restrictive. The scope of the invention is, therefore, indicated by the 
appended claims rather than by the foregoing description. All changes 
which come within the meaning and range of equivalency of the claims are 
to be embraced within their scope.