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BACKGROUND OF THE INVENTION
The present invention relates, in general, to a method and apparatus for
analysis of chemical species separated by differences in flow rate through
a porous medium, and, more particularly, to a method and apparatus for
analysis of such species through optical absorption, reflection,
refraction or fluorescence simultaneously in multiple micron-scale optical
channels. The invention further relates to a microfabricated porous medium
for such channels.
Optical systems for use in chemical analysis are well known, and the use of
such systems in electrophoretic DNA sequencing is a particularly important
application because of the intense interest in the sequencing of the human
genome. This is a multiyear, multibillion dollar project which is directed
to improving, if not revolutionizing, the ability to diagnose and treat
illness.
Significant progress is being made in this field, and commercial optical
systems are now available which are capable of sequencing DNA by gel
electrophoresis of fluorescently labeled DNA fragments.
Because DNA sequencing is a highly complex procedure which requires a great
deal of time and involves high cost, a considerable amount of research is
being done into techniques and devices for reducing the time required for
such sequencing, with one technique including the parallel reading of
fluorescence from multiple capillaries. However, problems still remain in
processing the DNA, in supplying it to the capillaries, in causing the DNA
to pass through the capillaries, and in optically reading out the results,
for currently available systems are relatively large, are expensive, and,
although capable of operating faster than previous systems, still require
very long time periods to sequence DNA fractions. Thus, the time required
to sequence the three billion base pairs which comprise the human genome
is still measured in years, and, there is an urgent need for an improved
optical system for carrying out such procedures. Such an improved system
would also have application in the analysis of other chemical species,
particularly where the species are separated by differences in flow rate
through a porous medium.
In electrophoretic analysis, chemical species are separated by an electric
field which produces varying flow rates, and the separated products may
then be detected optically. In typical DNA sequencing applications, the
DNA fragments to be analyzed are added to a gel material which carries the
fragments through electrophoresis channels. Such gels create problems,
however, since it is difficult to fill narrow capillaries with the gel
material, thereby increasing the time required and the expense of carrying
out the sequencing process. Efforts have been made to develop an
artificial gel material in the form of a porous medium, but the dimensions
of such structures have been limited to those obtainable by
photolithography. In addition, the production of artificial gel structures
by such a process is too expensive for practical use. Thus, there is a
need for an artificial gel structure which can be formed by processes that
are easily carried out over large areas and which can be fabricated in
inexpensive materials. Such a gel material would find wide use in a
miniaturized, compact, and proportionately less expensive systems for
chemical analysis.
SUMMARY OF THE INVENTION
Briefly, the present invention relates to a miniaturized optical system for
chemical analysis and to a microfabricated gel material usable in such a
miniaturized system. The invention utilizes multiple parallel microoptical
illumination paths from one or more light sources leading to individual
sample channels. The illumination from the light source may be divided and
directed in parallel to corresponding channels, or may be a single beam
scanned sequentially over the channels without a need for mechanically
translatable optics or the need for moving the sample channels.
Preferably, the illumination source is a laser.
Each optical path directs the illumination to a corresponding sample
channel where components of the sample material to be detected may be
caused to fluoresce, for example. Light emitted from the sample is
collected and focussed onto a detector array which may include individual
detectors for each sample channel, the detectors being responsive to
selected wavelengths in the emitted light for identification of the sample
material components. By using miniaturized optical components,
illumination and collection lenses and other optical elements can be
reduced to less than 1 mm diameter, and the illumination and collection
path length can be reduced to a total of about 1 cm. An array of aspheric
illumination and/or collection microlenses provides high optical
efficiency and further provides a complete set of optics for each of the
multiple sample channels.
By providing multiple sets of microptic illumination and collection paths
and multiple sample channels, the structure is completely scalable without
the need to extend the optical path length for any channel as the number
of sample channels increases. Further, the microoptical system of the
invention is easily mass produced by current commercial technologies, and
in large quantities can be produced relatively inexpensively.
Preferably, the optical system incorporates a carrier block which
incorporates a plurality of parallel sample channels. The carrier block
may be replicated in optically clear plastic from a master die, as by a
conventional molding process, to allow the sample channels to be readily
produced with precise dimensions at a low cost so as to provide disposable
sample holders. The block preferably is molded in two parts, one part,
which may be referred to as the channel block, including the parallel
sample channels on a first surface and the other part, which may be
referred to as the cover block, providing a cover for the channels. If
desired, channels or parts of channels, can be formed in both parts of the
carrier block, with the channels being completed and enclosed when the two
parts of the block are assembled.
In one form of the invention, one part of the carrier block, for example
the channel block, may incorporate multiple miniaturized illuminating
lenses on a second surface spaced from and parallel to the first surface
on which the sample channels are located. Preferably one lens is provided
for each channel, or for a small group of channels, for directing light
from an external source such as a laser (or multiple lasers) through the
channel block to the respective sample channels. The illuminating lenses
are molded as an integral part of the channel block in a preferred form of
the invention, although they may be adhesively secured to a surface of the
channel block, if desired.
In this embodiment, the second part of the carrier block, for example the
cover block, carries collection optics, preferably including a collection
lens for each channel for collecting output light passing through, emitted
by, or reflected from, sample material in the sample channels. The
collection optics also preferably include diffraction elements, to
separate the output light by wavelength, and directs this output light to
suitable detectors. The collection optics preferably are molded as an
integral part of the cover block, which also is optically clear plastic in
the preferred form of the invention, this fabrication process enabling
rapid and inexpensive replication of the cover block and optics.
The carrier block may incorporate suitable electrodes for supplying
electric potentials to sample material in the sample channels, to permit
electrophoretic analysis of this material. In this configuration the
structure of the present invention is particularly advantageous when used
in the fluorescent detection and analysis of sample material such as
dye-labeled DNA fragments in electrophoretic DNA sequencing. Accordingly,
the following description of preferred forms of the invention will be
particularly directed to this process, although it will be understood that
the described optical system lends itself to application in other
analytical processes.
The carrier block is illuminated by a suitable light source such as a
laser, although it may be utilized with other light sources such as solid
state laser arrays. The light is directed, in the foregoing form of the
invention, from a light source adjacent the second surface of the carrier
block through the illumination optics on that surface and through the
block to the sample channels. The light passes through the channels, with
some of the light striking the sample material in those channels to cause
fluorescence, for example. Fluorescent light emitted the first surface of
the carrier block is directed by the output optics on the cover block to
suitable detectors for measurement.
In another embodiment of the invention, the illumination and collection
optics are on the same side of the carrier block, and are separated by a
dichroic mirror. Illuminating light is directed onto the channels in the
carrier block by the dichroic mirror to cause fluorescence in the sample
material. Emitted fluorescence is directed back along the path of the
illuminating light and passes through the dichroic mirror to a detector in
this case, a single array of microlenses serves as both the illuminating
and the collection optics.
The sample channels may incorporate a porous material for separating a
sample material as it passes through the channel, for example under the
influence of an electric field in an electrophoresis process. In the
present invention, the porous material may be an artificial gel structure
incorporated in, and fabricated at the same time as, the sample channels.
The channels and the gel structure are fabricated by an etching process
which produces a very narrow channel and a multiplicity of micron-scale,
generally parallel, spaced pillars within this channel and perpendicular
to the direction of motion of sample material to be analyzed. As noted
above, the gel structure may be used as a die in a suitable glass or
plastic master mold, and replicated in a conventional molding process to
enable many copies to be made from a single mold by a simple, relatively
inexpensive process. The copies preferably are formed of optically clear
plastic.
When the above-described optical system is used in analysis of samples such
as DNA fragments, the carrier block is fabricated with multiple parallel
channels in the channel block portion, with the artificial gel structure
incorporated in all of the channels. The samples, such as dye-labeled DNA
fragments, are added to the channels and the channels are closed by the
second, cover block portion of the carrier block. Thereafter the carrier
block is placed between a laser light source and corresponding
fluorescence detectors and an electric field is provided in each channel
to separate the fragments in known manner. The illuminating light produces
characteristic fluorescence in the separated dye-labeled sample fragments,
so that parallel, and thus simultaneous, readings of the fluorescence of
the DNA fragments are obtained in each of the channels.
The carrier blocks are easily replicated in plastic, glass or other
transparent materials, and thus are inexpensive enough to allow
disposability of the blocks to prevent contamination between samples. The
small size also reduces the cost, and the geometry of the system allows
simultaneous readouts to increase the speed of analysis.
BRIEF DESCRIPTION OF THE DRAWINGS
The foregoing, and additional objects, features, and advantages of the
invention will be apparent to those skill in the art from the following
detailed description of a preferred embodiment thereof, taken in
conjunction with the accompanying drawings, in which:
FIG. 1 is a diagrammatic end view illustration of a miniaturized optical
chemical analysis system incorporating microfabricated flow channels, in
accordance with the present invention;
FIG. 2 is a diagrammatic top plan view of the system of FIG. 1,
illustrating in diagrammatic form plural parallel microfabricated flow
channels;
FIG. 3 is an exploded top plan view of a miniaturized optical system in
accordance with the present invention;
FIG. 4 is a partial top perspective view of the device of FIG. 3, partially
exploded;
FIG. 5 is a top plan view of a modified form of the optical analysis system
of the present invention;
FIG. 6 is a side elevation view of the system of FIG. 5;
FIG. 7 is an enlarged perspective view of a microfabricated flow channel
incorporating an artificial gel usable in the optical system of the
invention;
FIGS. 8-11 diagrammatically illustrate a process incorporating election
beam lithography for microfabrication of sample channels incorporating the
artificial gel of FIG. 7;
FIG. 12 is a scanning electron micrograph of an artificial gel test
structure fabricated by the process of FIGS. 8-11;
FIG. 13 is a scanning electron micrograph top view of an artificial gel
structure fabricated by a thin film mask and reactive ion etching;
FIG. 14 is a scanning electron micrograph of plural artificial gel
structures fabricated by electron beam exposure of a polymer; and
FIG. 15 illustrates closely spaced pillars incorporated as one wall of the
die mold.
DESCRIPTION OF PREFERRED EMBODIMENT
Turning now to a more detailed description of the present invention, FIGS.
1 and 2 illustrate in diagrammatic form a microlens array 10 for optical
analysis of sample material located in a plurality of elongated,
closely-spaced, generally parallel, coplanar sample channels 12, 14, 16
and 18. These channels may carry any desired chemical species for optical
analysis, but for illustration, the sample material is described herein as
including DNA fragments which are to be analyzed, or sequenced. The DNA
fragments, in one embodiment, may be carried by a gel material which is
injected into the respective sample channels. However, since currently
available gels limit the DNA sequencing process because of the difficulty
encountered in attempting to fill narrow capillary channels with the gel
material, in accordance with the preferred form of the present invention
the sample channels 12, 14, 16 and 18 are fabricated to include an
artificial gel material, as will be described below.
Although four sample channels are illustrated, it will be understood that a
large number of micro channels may be provided in order to improve the
throughput of the samples being analyzed. These channels may be located in
groups, may be evenly or irregularly spaced, as desired, and preferably
are generally linear and parallel to each other. When the analysis
involves DNA sequencing, the fragments are labeled with fluorescent dyes
in order to permit optical detection, as is known. These fragments are
fluoresced by impinging light from illumination laser beams 22, 24, 26 and
28 which are directed to corresponding sample channels by way of suitable
illumination optics such as illumination microlenses 32, 34, 36 and 38.
The laser beams are focused on the sample channels to cause the labeled
fragments to fluoresce at known wavelengths. This produces corresponding
output beams 42, 44, 46, and 48 which are directed by corresponding
collection lenses 52, 54, 56 and 58 through wavelength separation optics
such as diffraction gratings or prisms 62, 64, 66 and 68 which provide
wavelength separation of each output beam, as best illustrated in FIG. 1.
The separated wavelengths are directed to corresponding photo detectors
72, 74, 76 and 78, aligned with corresponding sample channels and their
respective collection lenses and gratings. Each photo detector comprises a
plurality of light sensitive elements such as the three elements 80, 82,
and 84 illustrated in FIG. 1 as photo included in detector 78.
Photodetectors 72, 74, 76 and 78 form an array of detector elements which
measure the output light from corresponding sample channels.
The illumination laser beams 22, 24, 26 and 28 may be generated by
corresponding individual solid state or similar lasers such as those
illustrated at 92, 94, 96 and 98, or may be from a single laser source
having its output beam divided into plural illumination beams.
The optical system of FIGS. 1 and 2 permits measurements of the optical
characteristics of a wide variety of sample materials. One example of such
a measurement is the parallel sequencing of DNA fragments, which is
carried out in the present device in a plurality of electrophoretic DNA
sample channels for improved speed of processing. In accordance with this
aspect of the present invention, a large number of very narrow sample
channels, in the range of 1 mm or less in width and depth, are fabricated
to incorporate a porous gel material which will receive the material to be
analyzed. A simple photolithographic process is used to define the
locations and dimensions of the channels, with the artificial gel material
described herein permitting the channels to have dimensions 10-100 times
smaller than was possible when prior conventional gel materials were used.
Of course, if such a conventional gel material is to be used in the
present optical system, the channels will be larger, but miniaturized
channels in combination with the artificial gel are preferred. The
preferred process for making the channels utilizes dry and isotropic ion
etches, rather than wet chemical etches, to form the channels and to
simultaneously fabricate the artificial gel within the channels.
An optical chemical analysis device incorporating the features described
above with respect to FIGS. 1 and 2 is illustrated generally at 100 in
FIGS. 3, 4, and 5, to which reference is now made. As there illustrated, a
two-component carrier block 102 is positioned between an illumination
source 104 and an output light detector array 106. The illumination source
104 incorporates the plural lasers 92, 94, 96, and 98, for example, while
the detector array 106 incorporates the photo detectors 72, 74, 76 and 78.
In a preferred form of the invention, the detector array 106 is a silicon
wafer which incorporates on one surface the light sensitive
photodetectors, with accompanying signal processing electronics being
embedded in the wafer. Alternatively, the detector elements may be
connected to suitable surface contact pads, such as those illustrated at
108, for connection to external detector circuitry.
The carrier block 102, in one embodiment of the invention, incorporates a
channel block 120 and a mating cover block 122, the channel block
containing a large number of miniature parallel sample channels, such as
the channels 12, 14, 16 and 18 previously described, fabricated on a
planar inner side wall 124. The cover block 122 includes a planar inner
side wall 126 opposed to, or facing, the wall 124, with the walls 124 and
126 engaging each other, as illustrated in FIG. 4, when the component
blocks 120 and 122 are mated together, so that the cover 122 closes the
channels 12, 14, 16, and 18. The cover 122 may be provided with alignment
ribs, such as the ribs 128 and 130, which engage corresponding alignment
grooves 132 and 134 on the face 124 of channel block 120 to ensure proper
alignment of component blocks 120 and 122 when the carrier block 102 is
assembled by mating the two components.
In one form of the invention, the blocks 120 and 122 are made of plastic,
glass, quartz, or similar optically clear, transparent material. In the
illustrated embodiment, each of the blocks 120 and 122 are generally
rectangular, with carrier block 120 having a planar outer side wall 134
generally parallel to inner wall 124 and having top and bottom walls 136
and 138. Similarly, cover block 122 includes an outer side wall 140
parallel to inner side wall 126 and further includes top and bottom walls
142 and 144. Channel block 120 preferably incorporates a plurality of
lenses, such as the lenses 32, 34, 36 and 38 illustrated diagrammatically
in FIG. 2, with one lens provided for each sample channel. These lenses
preferably are integrally formed microlenses fabricated in an array 146 on
the block 120, as illustrated at 32', 34', 36', and 38' in FIGS. 3 and 4,
although it will be understood that individual lenses may be secured to
the outer side wall 134, as by an adhesive, or mounted close to wall 134
on a separate holder, if desired. The lenses are optically aligned with
respective sample channels 12, 14, 16, and 18 so that sample illuminating
light from each of the laser sources 92, 94, 96 and 98 is directed by its
corresponding lens to a corresponding sample channel or to a corresponding
group of channels.
The cover block 122 also incorporates a plurality of lenses such as the
collection lenses 52, 54, 56 and 58 illustrated diagrammatically in FIG.
2. These lenses preferably are integrally formed microlenses fabricated in
an array 148 on the block 122 as illustrated at 52', 54', 56', and 58' in
FIG. 3, although it will be understood that separate lenses may be secured
to the outer side wall 140 of block 122 as by an adhesive, or may be
mounted on a suitable holder close to side wall 140, if desired. In a
preferred form of the invention, the collection lenses 52', 54', 56' and
58' each incorporate a diffraction grating which divides the output light
from each of the sample channels into its separate wavelength components.
The collection lenses then direct the different wavelengths of the output
light from each sample channel to corresponding detector elements of the
detectors 72, 74, 76, and 78 of detector array 106. If desired, a separate
diffraction grating or prism can be incorporated between the collection
lens array 148 and the detector to provide the required color separation.
It will be understood that the channel block component 120 and the cover
block component 122 can be fabricated in a variety of ways. Preferably,
however, both of the mating carrier block components are molded to
incorporate on outer surfaces 134 and 140 their respective illumination
lens array 146 and collection lens array 148, with the inner side wall 124
of block 120 being molded to incorporate the parallel sample channels 12,
14, 16 and 18 and the alignment grooves 132 and 134, and wall 126 of block
122 being molded to incorporate mating ribs 128 and 130. A sample to be
analyzed, such as a gel material containing DNA fractions, may then be
placed in the sample channel and the cover block mated to the channel
block to close the channels. Alternatively, the cover block 122 can be
mated to the channel block 120 and the sample material injected into the
individual channels. As a further modification, matching channels can be
provided on both the channel block and the cover block, with each block
then providing a portion of the sample channel depth.
The fabrication of blocks 120 and 122 with miniaturized sample channels and
unitary illumination and collection microlenses is carried out using a
conventional molding process wherein a two-part die (for example) receives
a flowable, optically clear material such as plastic or glass. The die is
shaped to define the microlenses, the diffraction grating (if it is to be
a unitary part of the collection optics), the sample channels and the
mating alignment ribs and grooves. By using a molding process to form the
lens arrays and sample channels unitarily, the cost of replicating the
system can be significantly reduced.
When the present system is utilized for electrophoretic analysis of a
sample, suitable electrodes are provided, for example, on the surface 126
of the cover 122, for contact with the sample material in the channels.
Such electrodes are illustrated in FIG. 4 at 150, 152, 154 and 156 as
being aligned with the tops of the respective channels 12, 14, 16 and 18,
and it will be understood that similar electrodes will be provided at the
bottom of each channel. These electrodes are connected to suitable voltage
sources as by contacts engaging the electrodes through the respective
sample channels.
In electrophoresis of DNA fragments utilizing the optical system of the
invention, the DNA fragments are fluorescently labeled before they are
placed in the sample channels. A suitable voltage is applied between the
top electrodes 150, 152, 154, 156 and corresponding bottom electrodes (not
shown) for each channel to produce electrophoretic flow of the sample
material in the channels to separate the fragments. Laser light is then
directed through the illumination lens array 146 and through block 120 to
the respective channels containing the fluorescently labeled DNA
fragments, causing the dye to fluoresce. Output fluorescent light from
these fragments passes through block 122, is collected by the collection
lens array 148 and is directed toward the detector array 106. The various
wavelengths of light corresponding to the fluorescent dye outputs are
separated by the diffraction gratings or prisms in the collection optics.
As previously described, the diffraction gratings can be either separate
elements, as illustrated in FIG. 2, or can be a part of the collection
lens structure, as illustrated in FIG. 3. The respective wavelengths are
directed to corresponding detector elements on detector array 106.
The dimensions of the microlens array of FIGS. 3 and 4 can be extremely
small. Each sample channel can have a width w and a depth d, illustrated
in FIG. 3 for channel 12, in the range of between about 1 .mu.m and 1 mm.
The corresponding illumination lens on the channel block for each channel
may have a diameter on the same order of magnitude; i.e, less than 1 mm,
to allow a spacing of about 1 mm between adjacent channels. Similarly,
each of the collection lenses carried by the cover 122 has a diameter of
less than about 1 mm. This permits a large number of channels with
corresponding illumination and collection optics to be placed on a
relatively small carrier block to provide a large number of parallel
outputs and a significant increase in the speed of the sequencing process.
Since each of the channels preferably has its own illumination optics, its
own collection optics and its own detector, simultaneous readout of the
channels is enabled without the need for scanning the laser source and/or
the detector.
A modification of the optical system of the invention is illustrated in
FIGS. 5 and 6, to which reference is now made. In this embodiment, a
modified two-component carrier block 168 is provided. This block includes
a channel block 170 having an inner planar wall 172 and an outer wall 174
generally parallel to wall 172. The channel block includes a top wall 176
and a bottom wall 178 which also are generally parallel to each other. The
inner wall 172 incorporates a plurality of sample channels 180, 182, 184,
and 186, for example, these channels being similar to the channels 12, 14,
16, and 18 described above and extending from the top wall 176 to the
bottom wall 178. Although illustrated as being regularly spaced, linear,
and generally parallel, these channels can be grouped or can be
irregularly spaced, can be curved, and need not be parallel, if desired.
An optically clear cover block 188 is secured to the inner surface 172 of
block 170 to close the channels in the manner described above with respect
to cover block 122. In this embodiment, however, neither the channel block
170 nor the cover block 188 incorporates a microlens array. Instead, a
microlens array 190 is provided on an inner surface 192 of an optically
clear lens block 194 which is spaced from cover block 188 and is generally
parallel to wall 172. A separate lens is preferably provided for each of
the sample channels, although a single lens can be used with a group of
channels, if desired. In the illustrated embodiment, individual lens
elements 200, 202, 204 and 206 are aligned with and correspond to sample
channels 180, 182, 184 and 186, respectively. The lens array 190 may be
integrally formed with the lens block 194 or may be adhesively secured to
the surface 192 thereof. Preferably, the block 194 is of glass or plastic
and the lenses are integrally molded as a part of the block. It will be
understood that, if desired, the cover 122 and the lens block 194 may be
combined with the lens array 190 located on the outer surface of the cover
in the manner illustrated for array 148 in FIG. 3.
The lens array 190 serves as the illumination lens for a sample material in
the channels. As discussed above, the sample may include fluorescent
dye-labeled DNA fragments, and the respective sample channels in channel
block 170 may incorporate electrodes such as electrode 150 illustrated in
FIG. 4 for use in electrophoretic analysis of the sample material. As
illustrated in the elevation view of FIG. 6, an illumination beam 208 is
directed to the respective channels from a laser source 210, the beam 208
being deflected by a surface 212 of a dichroic mirror 214 through the lens
block 194 and through the corresponding lenses such as lens 206 carried by
the block 194. The lenses focus the illumination onto their respective
electrophoresis channels, as illustrated by channel 186. The illuminating
light beam 208 causes the dye-labeled DNA fragments to fluoresce, with the
resulting output light 216 due to the fluorescence being collected by the
same lens array 190. The array directs the output light through lens block
194, through the dichroic mirror 214, and through a filter 218 to a
diffraction device 220 such as a grating or prism. The output light beam
216 is separated by wavelength at grating 220 and passes through a
cylindrical focussing lens 222 which directs the light onto corresponding
detector elements of detector array 224, which is similar to detector 106.
Laser illumination light which is reflected from the sample is indicated
by arrow 226. This light is deflected out of the system by dichroic mirror
214, so it does not interfere with the collection optics, but it can also
be measured, if desired, to determine the reflectivity of the sample.
In a preferred form of the invention, each of the sampl | | |
