Mesoscale sperm handling devices

Devices and methods are provided for the clinical analysis of a sperm sample. The devices comprise a solid substrate, typically on the order of a few millimeters thick and approximately 0.2 to 2.0 centimeters square, microfabricated to define a sample inlet port and a mesoscale flow channel extending from the inlet port. In one embodiment, a sperm sample is applied to the inlet port, and the competitive migration of the sperm sample through the mesoscale flow channel is detected to serve as an indicator of sperm motility. In another embodiment, the substrate of the device is microfabricated with a sperm inlet port, an egg nesting chamber, and an elongate mesoscale flow channel communicating between the egg nesting chamber and the inlet port. In this embodiment, a sperm sample is applied to the inlet port, and the sperm in the sample are permitted to competitively migrate from the inlet port through the channel to the egg nesting chamber, where in vitro fertilization occurs. The devices of the invention may be used in a wide range of applications in the analysis of a sperm sample, including the analysis of sperm morphology or motility, to assess sperm binding properties, and for in vitro fertilization.

REFERENCE TO RELATED APPLICATIONS 
This application is being filed contemporaneously with the following 
related copending applications: U.S. Ser. No. 07/877,702, filed May 1, 
1992; U.S. Ser. No. 07/877,701, filed May 1, 1992; U.S. Ser. No. 
07/877,536, filed May 1, 1992; and U.S. Ser. No. 07/877,662, filed May 1, 
1992, the disclosures of which are incorporated herein by reference. 
BACKGROUND OF THE INVENTION 
This invention relates generally to methods and apparatus for conducting 
analyses. More particularly, the invention relates to the design and 
construction of small, typically single-use, modules capable of rapidly 
analyzing microvolumes of a fluid sample. 
In recent decades the art has developed a very large number of protocols, 
test kits, and cartridges for conducting analyses on biological samples 
for various diagnostic and monitoring purposes. Immunoassays, 
agglutination assays, and analyses based on polymerase chain reaction, 
various ligand-receptor interactions, and differential migration of 
species in a complex sample all have been used to determine the presence 
or concentration of various biological compounds or contaminants, or the 
presence of particular cell types. 
Recently, small, disposable devices have been developed for handling 
biological samples and for conducting certain clinical tests. Shoji et al. 
reported the use of a miniature blood gas analyzer fabricated on a silicon 
wafer. Shoji et al., Sensors and Actuators, 15:101-107 (1988). Sato et al. 
reported a cell fusion technique using micromechanical silicon devices. 
Sato et al., Sensors and Actuators, A21-A23:948-953 (1990). Ciba Corning 
Diagnostics Corp. (USA) has manufactured a microprocessor-controlled laser 
photometer for detecting blood clotting. 
Micromachining technology originated in the microelectronics industry. 
Angell et al., Scientific American, 248:44-55 (1983). Micromachining 
technology has enabled the manufacture of microengineered devices having 
structural elements with minimal dimensions ranging from tens of microns 
(the dimensions of biological cells) to nanometers (the dimensions of some 
biological macromolecules). This scale is referred to herein as 
"mesoscale". Most experiments involving mesoscale structures have involved 
studies of micromechanics, i.e., mechanical motion and flow properties. 
The potential capability of mesoscale structures has not been exploited 
fully in the life sciences. 
Brunette (Exper. Cell Res., 167:203-217 (1986) and 164:11-26 (1986)) 
studied the behavior of fibroblasts and epithelial cells in grooves in 
silicon, titanium-coated polymers and the like. McCartney et al. (Cancer 
Res., 41:3046-3051 (1981)) examined the behavior of tumor cells in grooved 
plastic substrates. LaCelle (Blood Cells, 12:179-189 (1986)) studied 
leukocyte and erythrocyte flow in microcapillaries to gain insight into 
microcirculation. Hung and Weissman reported a study of fluid dynamics in 
micromachined channels, but did not produce data associated with an 
analytic device. Hung et al., Med. and Biol. Engineering, 9:237-245 
(1971); and Weissman et al., Am. Inst. Chem. Eng. J., 17:25-30 (1971). 
Columbus et al. utilized a sandwich composed of two orthogonally 
orientated v-grooved embossed sheets in the control of capillary flow of 
biological fluids to discrete ion-selective electrodes in an experimental 
multi-channel test device. Columbus et al., Clin. Chem., 33:1531-1537 
(1987). Masuda et al. and Washizu et al. have reported the use of a fluid 
flow chamber for the manipulation of cells (e.g. cell fusion). Masuda et 
al., Proceedings IEEE/IAS Meeting, pp. 1549-1553 (1987); and Washizu et 
al., Proceedings IEEE/IAS Meeting pp. 1735-1740 (1988). The art has not 
fully explored the potential of using mesoscale devices for the analyses 
of biological fluids and detection of microorganisms. 
The current analytical techniques utilized for the detection of 
microorganisms and cells are rarely automated, invariably employ visual 
and/or chemical methods to identify the strain or sub-species, and are 
inherently slow procedures. There is a need for convenient and rapid 
systems for clinical assays. There is particularly a growing need for 
standardized procedures for the analysis of semen, capable of providing 
reliable and rapid results, which may be used in the assessment of male 
infertility, and for a range of other applications including in vitro 
fertilization (IVF), artificial insemination by donor semen (AID) and 
forensic medicine. The World Health Organization, WHO Laboratory Manual 
for the Examination of Human Semen and Semen-Cervical Mucus Interaction, 
Cambridge University Press, Cambridge, U.K. (1987). The evaluation of male 
infertility through the analysis of semen involves a range of tests 
including the assessment of sperm count, motility, morphology, hormone 
levels, sperm antibodies, sperm cervical mucus interaction and sperm 
biochemistry. Wang et al., American Association for Clinical Chemistry, 
Endo. 10:9-15 (1992). There is a need for systems capable of conducting a 
range of rapid and reliable analyses of a sperm sample. 
An object of the invention is to provide analytical systems that can 
analyze microvolumes of a sperm sample and produce analytical results 
rapidly. Another object is to provide easily mass produced, disposable, 
small (e.g., less than 1 cc in volume) devices having mesoscale functional 
elements capable of rapid, automated analyses of sperm, in a range of 
applications. It is a further object of the invention to provide a family 
of such devices that individually can be used to implement a range of 
rapid tests, e.g., tests for sperm motility, and morphology. Another 
object is to provide a family of devices for conducting an in vitro 
fertilization in one device using microvolumes of sample. 
SUMMARY OF THE INVENTION 
The invention provides methods and apparatus for sperm handling. The 
devices may be used in a range of applications including sperm motility 
and morphology testing and in vitro fertilization. In one embodiment, the 
invention provides a device comprising a solid substrate, typically on the 
order of a few millimeters thick and approximately a 0.2 to 2.0 
centimeters square, microfabricated to define a sample inlet port and a 
mesoscale channel system. In one embodiment, the device may be used for in 
vitro fertilization. In this embodiment, the substrate of the device is 
micro-fabricated with a sperm inlet port, an egg nesting chamber, and an 
elongate mesoscale channel communicating between the egg nesting chamber 
and the inlet port, which permits competitive migration of sperm from the 
inlet port through the channel to the egg nesting chamber, where 
fertilization occurs. In another embodiment, the substrate may comprise a 
sperm inlet port and a mesoscale channel, extending from the inlet port. 
In this embodiment, sperm may be applied to the inlet port, and the extent 
of migration of the sperm through the channel can serve as an indicator of 
sperm motility or morphology. The term "mesoscale" is used herein to 
define flow passages having cross-sectional dimensions on the order of 
approximately 0.1 .mu.m to 500 .mu.m, with preferred widths on the order 
of 2.0 to 300 .mu.m, more preferably 3 to 100 .mu.m. For many 
applications, channels of 5-50 .mu.m widths will be useful. Chambers in 
the substrates often will have larger dimensions, e.g., a few millimeters. 
Preferred depths of channels and chambers are on the order of 0.1 to 100 
.mu.m, typically 2-50 .mu.m. 
In one embodiment, the mesoscale channel of the device may comprise a 
fractal region, comprising bifurcations leading to plural secondary 
channels, to enhance the detection or competitive migration of the sperm 
sample. The fractal region may comprise, equal numbers of bifurcations and 
junctions disposed serially along the direction of sperm migration. In one 
embodiment, the branching channels in the fractal region progressively 
decrease in cross-sectional area at each bifurcation and increase at each 
junction. The use of a mesoscale fractal flow channel is disclosed in U.S. 
Ser. No. 07/877,662, filed May 1, 1992, the disclosure of which is 
incorporated herein by reference. The devices and methods of the invention 
may be used to implement a variety of automated, sensitive and rapid, 
contaminant-free tests including clinical analyses of sperm properties and 
for rapid in vitro fertilization. 
Generally, as disclosed herein, the solid substrate comprises a chip 
containing the mesoscale channel and other functional elements. The 
channels and elements may be designed and microfabricated from silicon and 
other solid substrates using established micromachining methods. The 
chambers and channels in the devices may be microfabricated on the surface 
of the substrate, and then a cover, e.g., a transparent glass cover, may 
be adhered, e.g., anodically bonded over the surface. The devices 
typically are designed on a scale suitable to analyze microvolumes (&lt;10 
.mu.L) of sample, introduced into the flow system through an inlet port 
defined, e.g., by a hole communicating with the flow system through the 
substrate or cover slip. The volume of the mesoscale channels and chambers 
typically will be &lt;5 .mu.L, and the volumes of individual channels, 
chambers, or other functional elements are often less than 1 .mu.L, e.g., 
in the nanoliter or even picoliter range. Assays can be conducted rapidly 
and after an assay is complete, the devices can be discarded. 
The chips may be used with an appliance which contains a nesting site for 
holding the chip, and which mates one or more input ports on the chip with 
one or more flow lines in the appliance. Before or after a sperm sample is 
applied to the inlet port of the substrate, the chip may be placed in the 
appliance, and a pump, e.g., in the appliance, can be actuated to 
introduce a buffer or other fluid to hydraulically fill the channels and 
chambers or to force the sperm sample or other fluid components into (or 
out of) the flow system. Alternatively, sperm may be injected into the 
chip by the appliance. A sperm sample or other fluid component also may 
enter the channel system simply by capillary action through an inlet port. 
The fluid contents of the channels and chambers of the devices may be 
observed optically, either visually or by machine, through a translucent 
window, such as a transparent cover over the channel system, or through a 
translucent section of the substrate itself. Thus, the devices permit the 
optical detection, e.g., of sperm migration in a channel, or in another 
embodiment, egg fertilization in an egg nesting chamber. The appliance may 
comprise means for viewing the contents of the device such as an optical 
viewing system, such as a microscope or a camera. 
In another embodiment, the substrate of the device may include a sperm 
inlet port, a mesoscale channel extending from the inlet port, and a 
mesoscale detection chamber in fluid communication with the flow channel. 
The mesoscale detection chamber is provided with a binding moiety capable 
of binding with a preselected component of a sperm sample. The detection 
chamber may be provided, e.g., with a binding moiety capable of detectably 
binding to a sperm antibody or hormone, to enable the detection of a 
specific sperm component. 
The use of a detection region allows a range of binding assays to be 
performed on a sperm sample. The use of a mesoscale detection chamber is 
disclosed in U.S. Ser. No. 07/877,702, filed May 1, 1992. 
Some of the features and benefits of devices constructed in accordance with 
the teachings disclosed herein are summarized in Table 1. A device may 
include two or more separated systems, e.g., fed by a common inlet port, 
to implement a plurality of assays. The device may also comprise a control 
system so that data from the sample region and the control region may be 
detected and compared. The devices can be used to implement a range of 
rapid clinical tests for the analysis of a sperm sample. The devices may 
be utilized, e.g., for the detection of the motility or morphology of a 
sperm sample or to test the presence of sperm antibodies or hormones, or 
to test the interaction of sperm with cervical mucus, or other assays used 
in infertility testing. In addition, the devices may be utilized to test 
the interaction of a sperm sample with other reagents such as spermicides. 
The invention provides methods and devices for use in a wide range of 
possible assays. Assays may be completed rapidly, and at the conclusion of 
the assay the chip can be discarded, which advantageously prevents 
contamination between samples, entombs potentially biologically hazardous 
material, and provides an inexpensive, microsample analysis. 
TABLE 1 
______________________________________ 
Feature Benefit 
______________________________________ 
Flexibility No limits to the number of chip 
designs or applications available. 
Reproducible Allows reliable, standardized, mass 
production of chips. 
Low Cost Allows competitive pricing with 
Production existing systems. Disposable nature 
for single-use processes. 
Small Size No bulky instrumentation required. 
Lends itself to portable units and 
systems designed for use in non- 
conventional lab environments. 
Minimal storage and shipping costs. 
Microscale Minimal sample and reagent volumes 
required. Reduces reagent costs, 
especially for more expensive, 
specialized test procedures. Allows 
simplified instrumentation schemes. 
Sterility Chips can be sterilized for use in 
microbiological assays and other 
procedures requiring clean 
environments. 
Sealed System Minimizes biohazards. Ensures 
process integrity. 
Multiple Circuit 
Can perform multiple processes or 
Capabilities analyses on a single chip. Allows 
panel assays. 
Multiple Expands capabilities for assay and 
Detector process monitoring to virtually any 
Capabilities system. Allows broad range of 
applications. 
Reuseable Chips 
Reduces per process cost to the user 
for certain applications. 
______________________________________

DETAILED DESCRIPTION 
The invention provides methods and apparatus for sperm handling, which may 
be utilized in a range of applications including sperm motility and 
morphology testing and in vitro fertilization. The invention provides a 
device comprising a solid substrate, typically on the order of a few 
millimeters thick and approximately 0.2 to 2.0 centimeters square, 
microfabricated to define a sperm inlet port and a mesoscale flow system 
extending from the inlet port. In one embodiment, a sperm sample is 
applied to the inlet port and the extent of migration of the sperm through 
the channel can serve as an indication of, e.g., the motility or 
morphology of the sperm sample. In another embodiment, the substrate may 
further include an egg nesting chamber, and an elongate channel of 
mesoscale cross sectional dimension, communicating between the egg nesting 
chamber and the sperm inlet port. In operation, a sperm sample is applied 
to the inlet port, and sperm in the sample then migrate competitively 
through the channel to the egg chamber, where fertilization of the egg 
occurs. 
Analytical devices having mesoscale flow systems can be designed and 
fabricated in large quantities from a solid substrate material. They can 
be sterilized easily. Silicon is a preferred substrate material because of 
the well-developed technology permitting its precise and efficient 
fabrication, but other materials may be used including cast or molded 
polymers including polytetrafluoroethylenes. The sample inlet and other 
ports, the mesoscale flow system, including the flow channel(s) and other 
functional elements, may be fabricated inexpensively in large quantities 
from a silicon substrate by any of a variety of micromachining methods 
known to those skilled in the art. The micromachining methods available 
include film deposition processes such as spin coating and chemical vapor 
deposition, laser fabrication or photolithographic techniques such as UV 
or X-ray processes, or etching methods which may be performed by either 
wet chemical processes or plasma processes. (See, e.g., Manz et al., 
Trends in Analytical Chemistry, 10: 144-149 (1991)). 
Flow channels of varying widths and depths can be fabricated with mesoscale 
dimensions for use in analyzing a sperm sample. The silicon substrate 
containing a fabricated mesoscale flow channel may be covered and sealed, 
e.g., anodically bonded, with a thin glass cover. Other clear or opaque 
cover materials may be used. Alternatively, two silicon substrates can be 
sandwiched, or a silicon substrate can be sandwiched between two glass 
covers. The use of a transparent cover results in a window which 
facilitates dynamic viewing of the channel contents, and allows optical 
probing of the mesoscale flow system either visually or by machine. Other 
fabrication approaches may be used. 
The capacity of the devices is very small and therefore the amount of 
sample fluid required for an analysis is low. For example, in a 1 
cm.times.1 cm silicon substrate, having on its surface an array of 500 
grooves which are 10 microns wide.times.10 microns deep.times.1 cm 
(10.sup.4 microns) long, the volume of each groove is 10.sup.-3 .mu.L and 
the total volume of the 500 grooves is 0.5 .mu.L. The low volume of the 
mesoscale flow systems allows assays to be performed on very small amounts 
of a liquid sample (&lt;5 .mu.L). The mesoscale devices may be 
microfabricated with microliter volumes, or alternatively nanoliter 
volumes or less, which advantageously limits the amount of sample, buffer 
or other fluids required for an analysis. Thus, an important consequence 
and advantage of employing flow channels having mesoscale dimensions is 
that very small scale analyses can be performed. 
In one embodiment, illustrated schematically in FIGS. 1, 2 and 3, the 
device 10 may be utilized for a rapid assay of, e.g., the motility or 
morphology of a sperm sample. Device 10 includes a silicon substrate 14 
microfabricated with ports 16, primary sample channel 20A, and a fractal 
system of channels 40. The ports may be microfabricated with mesoscale or 
larger dimensions. The fractal region 40 in this case comprises equal 
numbers of bifurcations and junctions, disposed serially through the 
fractal region, leading to a third channel 20B. The substrate 14 is 
covered with a clear glass or plastic window 12 to form an enclosing wall 
of the channels. In operation, after hydraulically filling all channels 
with an appropriate liquid sperm medium, e.g., cervical mucus or a buffer, 
a sperm sample is applied at inlet port 16A. Sperm in the sample migrate 
into flow channel 20A, and then through the fractal region 40 towards 
channel 20B and port 16B. The extent of progress of a sperm sample through 
the fractal path 40 can serve as an indicator of sperm motility and 
morphology. The flow of a sperm sample may be detected optically, e.g., 
with a microscope, either visually or by machine, through the transparent 
cover over the flow system, or through a transparent region of the 
substrate itself. 
In another embodiment, the fractal system 40 may be microfabricated on a 
silicon substrate with reduced dimensions at each bifurcation, providing 
sequentially narrower flow channels, as illustrated schematically in FIG. 
5. FIG. 5 shows device 10, which comprises substrate 14 microfabricated 
with fractal flow channels 40, which have a reduced cross-sectional area 
relative to the primary flow channel 20A and the third flow channel 20B. 
In operation, sperm in a sample enter to the device 10 through inlet port 
16A and channel 20A, and migrate through the fractal region 40 towards 
channel 20B and port 16B. The fractal region 40 provides an extensive 
network suitable for the competitive migration of a sperm sample. The 
fractal system may be microfabricated with a more complex series of 
bifurcations, as illustrated schematically in device 10 in FIG. 10, which 
includes a pair of fractally bifurcating channels 40A and 40B. The fractal 
channel network 40A is constructed with sequentially narrower channels 
towards the center of the fractal, thereby enhancing sensitivity to sperm 
migration. 
The analytical devices containing the mesoscale channel system can be used 
in combination with an appliance for delivering and receiving fluids to 
and from the devices, such as appliance 50 shown schematically in FIG. 4, 
which incorporates a nesting site 58 for holding the device 10, and for 
registering ports, e.g., ports 16 on the device 10, with a flow line 56 in 
the appliance. The appliance also includes pump 52 which may be used to 
inject or receive sample fluids into or from device 10. Alternatively, the 
sample may be injected into the device, or may enter the flow system 
simply by capillary action. Devices such as valves and other mechanical 
sensors for detecting sample fluid in the devices can be fabricated 
directly on the silicon substrate and can be mass-produced according to 
well established technologies. Angell et al., Scientific American, 
248:44-55 (1983). Alternatively, sensors such as optical detectors and 
other detection means may be provided in the appliance utilized in 
combination with the device. 
In one embodiment, the analytical devices also may be utilized in 
combination with an appliance for viewing the contents of the devices. The 
appliance may comprise a microscope for viewing the contents of the 
chambers and channels in the devices. In another embodiment, a camera may 
be included in the appliance, as illustrated in the appliance 60 shown 
schematically in FIGS. 11 and 12. The appliance 60 is provided with a 
housing 62, a viewing screen 64 and a slot 66 for inserting a chip into 
the appliance. As shown in cross section in FIG. 12, the appliance 60 may 
also include a video camera 68, an optical system 70, and a tilt mechanism 
72 for holding device 10, and allowing the placement and angle of device 
10 to be adjusted manually. The optical system 70 may include a lens 
system for magnifying the channel contents, as well as a light source. The 
video camera 68 and screen 64 allow sample fluids to be monitored 
visually, and optionally to be recorded using the appliance. 
In another embodiment, the substrate may be disposed, e.g., in an 
appliance, at an angle with respect to a horizontal plane, to provide an 
incline for the travel of a sperm sample, to further enhance the detection 
of motility. In another embodiment, the sperm flow channel may comprise 
protrusions 122, illustrated in FIG. 8, to provide a barrier for 
competitive migration of sperm. 
The devices may be microfabricated with a mesoscale flow channel that 
includes a detection region for detecting a component of a sperm sample, 
such as sperm antibodies or hormones. The detection region may comprise a 
binding moiety, capable of binding to a predetermined component of the 
sperm sample. The binding moiety, such as an antigen binding protein, may 
be immobilized on the surface of the flow channels, or on a solid phase 
reactant such as a bead. The binding moiety in the detection region may be 
introduced into the detection region in solution, or alternatively, may be 
immobilized on the surface of the mesoscale flow channels by, e.g., 
physical absorption onto the channel surfaces, or by chemical activation 
of the surface and subsequent attachment of biomolecules to the activated 
surface. Techniques available in the art may be utilized for the chemical 
activation of silaceous channel surfaces, and for the subsequent 
attachment of a binding moiety to the surfaces. (See, e.g., Haller in: 
Solid Phase Biochemistry, W. H. Scouten, Ed., John Wiley, New York, pp 
535-597 (1983); and Mandenius et al., Anal. Biochem., 137:106-114 (1984), 
and Anal. Biochem., 170:68-72 (1988)). The use of a binding moiety for 
assays in a mesoscale detection chamber, as well as techniques for 
providing the binding moiety in the detection chamber, are disclosed in 
the copending related application, U.S. Ser. No. 07/877,702, filed May 1, 
1992. The detection chamber may be utilized in a range of binding assays, 
e.g., to assay the interaction of a sperm sample with cervical mucus, to 
test the efficacy of spermicides, to assay for the presence of antibodies 
or contaminants in the sample, or to conduct sperm counts. 
In one embodiment, the binding moiety may be immobilized on a particle 
capable of inducing detectable agglomeration of a component of a sperm 
sample in a fractal mesoscale flow system. As illustrated in device 10, 
shown schematically in FIG. 6, particles 42, coated with binding protein 
specific for a given analyte in the sperm sample, may be provided in the 
fractal region 40 to promote analyte-induced agglomeration of fluid in the 
fractal region. Agglomeration in the fractal region may be detected 
optically through a window, e.g., disposed over the fractal region, or, 
e.g., by detecting pressure or conductivity changes of the sample fluid. 
In another embodiment, the devices of the invention may be utilized to 
conduct an in vitro fertilization. One embodiment of an in vitro 
fertilization device is shown in FIG. 7. Device 10 in FIG. 7 includes a 
sperm chamber 22C and an egg nesting chamber 22D, connected by a mesoscale 
fractal channel system 40. The device includes a clear cover 12, which is 
disposed over the fractal region and partly across the top of chambers 22C 
and 22D, leaving an open port at the top of the chambers. Alternatively, 
the cover 12 may extend over the entirety of the surface (not shown), and 
define holes disposed over the chamber 22C and 22D, which permit 
introduction of sperm and egg, but discourage evaporation. In operation, a 
sperm sample is applied to chamber 22C, e.g., through the top of the 
chamber. An egg is placed in the nesting chamber 22D. The flow system 
including chambers 22C and 22D, channels 20 and the fractal region 40, are 
provided with a buffer including, e.g., mammalian tubal fluid. The flow 
system also can include the buffer chambers 22B and 22A, in fluid 
communication with the flow system, which are filled with buffer to 
alleviate the potentially destructive effects of fluid loss from 
evaporation or dehydration from within the substrate. Competitive 
migration of the sperm sample from chamber 22C occurs through the fractal 
region 40 to the egg nesting chamber 22D where fertilization of the egg 
occurs. Fertilization can be determined, e.g., optically, either visually 
or by machine, by observing early stages of egg cell division. The device 
may be utilized in combination with an appliance mated to ports in the 
device for the addition or withdrawal of fluid components from the device. 
The appliance may include, e.g., means, such as a pump or syringe, for 
hydraulically expelling a fertilized egg from the device subsequent to 
fertilization, e.g., directly into a host uterus, e.g., by forcing saline 
or other liquid through the channels. 
The mesoscale channel system may be microfabricated with a filter for 
filtering sperm sample components. The filter may be microfabricated in 
the flow system between the sperm inlet port and the egg nesting region to 
enable the filtration of the sample. Filters which may be microfabricated 
in the flow system include the filters 24 shown in FIGS. 13, 14 and 15. In 
the device 10, the Filter 24 is microfabricated between the flow channels 
20A and 20B allowing sample fluid in channel 20A to pass through the 
filter 24. The filtrate exits through the filter 24 into channel 20B. 
Filter 24 comprises mesoscale flow channels of reduced diameter in 
comparison with channel 20A-20B, microfabricated with depths and widths on 
the order of 0.1 to 20 .mu.m. In contrast, the flow channels 20A and 20B 
have widths on the order of a maximum of approximately 500 .mu.m and more 
typically 100 .mu.m. Other filter means may be utilized, such as the posts 
122 extending from a wall of the flow channel 20 shown in FIG. 8. 
The devices may be used to implement a variety of automated, sensitive and 
rapid clinical analyses of a sperm sample. The devices can be used in a 
range of applications including fertility tests of a sperm sample, tests 
of sperm binding properties, in vitro fertilization, and forensic 
analyses. In order to enhance the accuracy of an assay, the substrate may 
be fabricated to include a control region in the flow system, e.g., a 
region which is identical in geometry to the test region, but does not 
include binding moieties. Sample fluid is directed to both the analytical 
and control regions to allow the comparison of the regions. The devices 
also may comprise a plurality of mesoscale flow systems to enable a 
plurality of assays to be conducted on a sperm sample. At the conclusion 
of the assay the devices typically are discarded. The use of disposable 
devices eliminates contamination among samples. The sample at all times 
can remain entombed, and the low volume simplifies waste disposal. 
The invention will be understood further from the following nonlimiting 
examples. 
EXAMPLE 1 
Sperm motility is tested in the chip 10 shown schematically in FIG. 5. A 
sample of semen (&lt;2 .mu.L) is placed on a glass microscope slide, and the 
chip 10 is placed on top of the semen sample such that the port 16A is 
positioned on the semen sample. The progress of individual spermatozoa 
into port 16A, through channel 20A and fractal region 40 is monitored 
using a microscope. The experimental results may be compared with results 
previously established for a healthy sperm sample to provide a test of 
sperm motility. 
EXAMPLE 2 
A channel containing a barrier 122 with 7 .mu.m gaps (illustrated in cross 
section in FIG. 8) is filled with HTF-BSA medium and a semen sample 
applied at the entry hole. The progression of the sperm through the 
barrier serves as an indicator of sperm motility. 
EXAMPLE 3 
Sperm functions are tested on the microfabricated solid substrate 14 shown 
in FIG. 9. A sperm sample is added to the inlet port 16A and then flows 
through the mesoscale flow channel 20 to the detection chambers 40A, 40B 
and 40C. Fractal detection chamber 40A provides a test for leucocytes and 
comprises immobilized antibody to common leukocyte antigen. Fractal 
detection chamber 40B provides a test for sperm antibodies and contains 
immobilized antibody to human IgG, IgA or IgM. Fractal detection chamber 
40C provides a test for acrosome reaction and contains fluorescein labeled 
lectin. Flow restriction due to agglutination in the chambers may be 
detected, e.g., by optical detection through a glass cover disposed over 
the substrate. After the assay is complete, the device is discarded. 
EXAMPLE 4 
A chip of the type illustrated in FIG. 7, defining an egg nesting chamber 
and a sperm inlet port, connected by a mesoscale channel, was washed with 
ultra-pure water and then filled with HTF-BSA. Eggs and semen were 
harvested from appropriate donors. A single egg was transferred to the egg 
nesting chamber using a micropipette, and a sample of semen was applied to 
the sperm inlet port using a micropipette. This entire procedure was 
conducted under a laminar flow hood and the application of the egg and 
semen was confirmed visually using a microscope. Progression (and 
selection of sperm) through the flow channel connecting the sperm inlet 
port and the egg nesting chamber containing the egg was confirmed 
visually. The chip was placed in a moist environment to minimize 
evaporation from the chip, and then incubated at 37.degree. C. for several 
hours. Fertilization of the egg was confirmed by visual inspection. 
Implantation of the fertilized egg was achieved by expelling the entire 
contents of the chip. Additionally, the chip contains a reservoir of 
HTF-BSA in connection with the chambers and flow channel in order to 
compensate for any evaporation from the chip. 
EXAMPLE 5 
Experiments were performed in mesoscale flow channels testing the sperm 
motility of human semen samples. In a sperm motility test, microchannels 
(80 .mu.m wide, 20 .mu.m deep, and 10 mm long) in a glass-silicon chip 
were filled with Human Tubal Fluid (HTF) medium (Irvine Scientific, Santa 
Ana, Calif.) containing 0.5% BSA (HTF-BSA). A sample of semen (&lt;2 .mu.L) 
was placed on a glass microscope slide and the chip placed on top of the 
semen sample such that the entrance to the channel was positioned on the 
semen sample. The progress of individual spermatozoa into the channel and 
along its length to the exit hole was monitored using a microscope, and 
recorded using a TV camera and video recorder. Sperm were observed 
traversing the entire length of the channel and could be seen accumulating 
in the exit hole. Migration of sperm was also demonstrated in channels of 
40, 100, and 120 .mu.m depths. 
Sperm motility in fractal channels also was determined, by examining the 
distance the sperm traveled along the fractal flow path. The above 
experiment was repeated using a fractal channel (40 .mu.m wide, 20 .mu.m 
deep) filled with HTF-BSA medium. Sperm were observed migrating through 
the tortuous fractal pathway (a total of 9 right angle turns, e.g., the 
device of FIG. 11) from the entry to the center of the channel. The 
experiment was repeated using a fractal channel which was 20 .mu.m deep, 
but which was reduced in width at each bifurcation (40, 30, 25, 20, and 10 
.mu.m) and then increased in width (20, 25, 30, 40 .mu.m). Again sperm 
migrated to the center of the fractal channel. 
The bi-directional motility of a sperm sample was also examined. A channel 
(60 and 80 .mu.m wide, 20 .mu.m deep) and fractal channels were filled 
with HTF-BSA medium and semen introduced simultaneously via the holes at 
each end of the channel. Sperm were observed migrating towards the center 
of the channel (or fractal channel) and eventually passing as they 
migrated towards the hole at the opposite end of the channel. 
An inclined channel experiment was also performed on a sperm sample. A 
channel (60 .mu.m wide, 20 .mu.m deep) was filled with HTF-BSA medium and 
a sample of sperm applied to the inlet hole. The inlet and outlet holes 
were sealed with adhesive tape. The chip was inclined at 45.degree. for 
different periods of time and then the progression of the sperm up the 
channel determined visually. Sperm were found to migrate efficiently up 
the inclined channel and could be seen in the exit hole at the top of the 
channel. 
EXAMPLE 6 
An experiment testing different spermicides using a mesoscale flow system 
was conducted. A chip comprising two chambers (5.2 mm long, 750 .mu.m 
wide, 1.5 mm deep) each linked at each end to an entry hole by a channel 
(3.25 mm long, 100 .mu.m wide, 20 .mu.m deep) was used for the 
simultaneous testing of the spermicidal activity of nonoxynol-9 and C13-G 
(Biosyn, Inc., Pa.). The four channels were filled with HTF-BSA solution 
(channel #1, control), 0.005% (channel #2), 0.0125% (channel #3), and 
0.05% (channel #4) nonoxynol-9 (or C13-G), respectively. A sample of semen 
was placed in each chamber and the progress of sperm into the adjoining 
channels monitored using the microscope. The number of sperm observed in 
the channels was in the following order of decreasing sperm count: channel 
#1&gt; #2&gt; #3&gt; #4. Most sperm were seen in the control channel, and none were 
seen in channel #4 which contained nonoxynol-9 or C13G at the optimum 
concentration for spermicidal action. 
EXAMPLE 7 
A morphological examination of motile sperm was conducted in a mesoscale 
flow system. A chip comprising two chambers (5.2 mm long, 750 .mu.m wide, 
1.5 mm deep) each linked at each end to an entry hole by a channel (3.25 
mm long, 100 .mu.m wide, 20 .mu.m deep) was used. The channels were filled 
with HTF-BSA solution and a semen sample applied to the central chamber. 
The chip was placed in a moist environment for 10 minutes. The surface 
solution from the holes at each end of the chip was removed and placed on 
a glass microscope slide (previously washed with ethanol). The slide was 
dried at 40.degree. C. then stained using Wright Giemsa stain (Curtin 
Matheson Scientific, Inc., Houston, Tex.). The sperm which had migrated 
from the central chamber to the end of the channel and into the hole had a 
normal morphological appearance. 
EXAMPLE 8 
The interaction of a sperm sample with cervical mucus in a mesoscale flow 
system was tested in a chip comprising two chambers (5.2 mm long, 750 
.mu.m wide, 1.5 mm deep) each linked at each end to an entry hole by a 
channel (3.25 mm long, 100 .mu.m wide, 20 .mu.m deep). The channels were 
filled with HTF-BSA solution and a cervical mucus sample (collected at 
approximately day 14 of the patient's menstrual cycle) placed in each of 
the central chambers. Sperm did not migrate into the cervical mucus and 
those that penetrated died, as anticipated because cervical mucus is known 
to be hostile to sperm at this time during the menstrual cycle. Moghissi 
et al., Am. J. Obstet. Gynecol., 114:405 (1972). 
EXAMPLE 9 
A test of the interaction of hyaluronic acid with a sperm sample was 
conducted to assess the cervical interaction of a sperm sample. The test 
was conducted in a chip comprising two chambers (5.2 mm long, 750 .mu.m 
wide, 1.5 mm deep) each linked at each end to an entry hole by mesoscale 
flow Channels #1, #2, #3 and #4 (3.25 mm long, 100 .mu.m wide, 20 .mu.m 
deep). Channel #1 was a control channel. Channels were filled with HTF-BSA 
solution and solutions of hyaluronic acid (Sigma) in HTF-BSA (channels #2, 
#3, #4, 5 mg/mL, 2.5 mg/mL, and 1.3 mg/mL, respectively). A semen sample 
was placed in each of the central chambers. Sperm did not migrate into 
channel #2, containing 5 mg/mL hyaluronic acid, but the extent of 
migration increased as the concentration of hyaluronic acid decreased in 
channels #3 and #4. 
EXAMPLE 10 
An immunobead test for the presence of IgG antibodies in a sperm sample was 
conducted. Immunobeads (BioRAD, Richmond, Calif.), microbeads coated with 
an antibody to human IgG, were diluted to 1 mg/mL in HTF-BSA solution 
(Irvine Scientific, Santa Ana, Calif.). A microchannel (250 .mu.m wide, 20 
.mu.m deep, and 10 mm long) in a glass-silicon chip was filled with a 
sample of the immunobead solution and a semen sample (ca 1.2 .mu.L) was 
applied to the channel entry. Agglutination of sperm by the immunobeads 
due to the presence of antibodies in the sperm sample was observed in the 
channel. As a control, the experiment was performed on a glass microscope 
slide using larger volumes of the immunobead reagent and semen sample, and 
this was also positive (agglutination observed). 
It will be understood that the above descriptions are made by way of 
illustration, and that the invention may take other forms within the 
spirit of the structures and methods described herein. Variations and 
modifications will occur to those skilled in the art, and all such 
variations and modifications are considered to be part of the invention, 
as defined in the claims.