US20040106130A1 - Bioarray chip reaction apparatus and its manufacture - Google Patents

Abstract

A body300having a cavity310for mounting a substrate120fabricated with probe sequences at known locations according to the methods disclosed in U.S. Pat. No. 5,143,854 and PCT WO 92/10092 or others, is provided. The cavity includes inlets350and360for introducing selected fluids into the cavity to contact the probes. Accordingly, a commercially feasible device for use in high throughput assay systems is provided.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS
• This application is a continuation of U.S. application Ser. No. 10/229,759, filed on Aug. 28, 2002, which is a continuation of U.S. patent application Ser. No. 10/046,623, filed Jan. 14, 2002, now U.S. Pat. No. 6,551,817, which is a continuation of U.S. patent application Ser. No. 09/907,196, filed Jul. 17, 2001, now U.S. Pat. No. 6,399,365, which is a continuation of U.S. patent application Ser. No. 09/302,052, filed Apr. 29, 1999, now U.S. Pat. No. 6,287,850, which is a continuation of U.S. patent application Ser. No. 08/485,452, filed Jun. 7, 1995, now U.S. Pat. No. 5,945,334, which is continuation-in-part U.S. patent application Ser. No. 08/255,682, filed Jun. 8, 1994, now U.S. Pat. No. 6,140,044. Each of these applications is incorporated herein by reference in its entirety for all purposes.[0001]
BACKGROUND OF THE INVENTION
• The present inventions relate to the fabrication and placement of materials at known locations on a substrate. In particular, one embodiment of the invention provides a method and associated apparatus for packaging a substrate having diverse sequences at known locations on its surface.[0002]
• Techniques for forming sequences on a substrate are known. For example, the sequences may be formed according to the pioneering techniques disclosed in U.S. Pat. No. 5,143,854 (Pirrung et al.), PCT WO 92/10092, or U.S. application Ser. No. 08/249,188, now U.S. Pat. No. 5,571,639, incorporated herein by reference for all purposes. The prepared substrates will have a wide range of applications. For example, the substrates may be used for understanding the structure-activity relationship between different materials or determining the sequence of an unknown material. The sequence of such unknown material may be determined by, for example, a process known as sequencing by hybridization. In one method of sequencing by hybridization, a sequences of diverse materials are formed at known locations on the surface of a substrate. A solution containing one or more targets to be sequenced is applied to the surface of the substrate. The targets will bind or hybridize with only complementary sequences on the substrate.[0003]
• The locations at which hybridization occurs can be detected with appropriate detection systems by labeling the targets with a fluorescent dye, radioactive isotope, enzyme, or other marker. Exemplary systems are described in U.S. Pat. No. 5,143,854 (Pirrung et al.) and U.S. patent application Ser. No. 08/143,312, also incorporated herein by reference for all purposes. Information regarding target sequences can be extracted from the data obtained by such detection systems.[0004]
• By combining various available technologies, such as photolithography and fabrication techniques, substantial progress has been made in the fabrication and placement of diverse materials on a substrate. For example, thousands of different sequences may be fabricated on a single substrate of about 1.28 cm[0005]²in only a small fraction of the time required by conventional methods. Such improvements make these substrates practical for use in various applications, such as biomedical research, clinical diagnostics, and other industrial markets, as well as the emerging field of genomics, which focuses on determining the relationship between genetic sequences and human physiology.
• As commercialization of such substrates becomes widespread, an economically feasible and high-throughput device and method for packaging the substrates are desired.[0006]
SUMMARY OF THE INVENTION
• Methods and devices for packaging a substrate having an array of probes fabricated on its surface are disclosed. In some embodiments, a body containing a cavity is provided. A substrate having an array of probes is attached to the cavity using, for example, an adhesive. The body includes inlets that allow fluids into and through the cavity. A seal is provided for each inlet to retain the fluid within the cavity. An opening is formed below the cavity to receive a temperature controller for controlling the temperature in the cavity. By forming a sealed thermostatically controlled chamber in which fluids can easily be introduced, a practical medium for sequencing by hybridization is provided.[0007]
• In other embodiments, the body is formed by acoustically welding two pieces together. The concept of assembling the body from two pieces is advantageous. For example, the various features of the package (i.e., the channels, sealing means, and orientation means) are formed without requiring complex machining or designing. Thus, the packages are produced at a relatively low cost.[0008]
• In connection with one aspect of the invention, a method for making the chip package is disclosed. In particular, the method comprises the steps of first forming a plurality of probe arrays on a substrate and separating the substrate into a plurality of chips. Typically, each chip contains at least one probe array. A chip is then mated to a package having a reaction chamber with fluid inlets. When mated, the probe array is in fluid communication with the reaction chamber.[0009]
• In a specific embodiment, the present invention provides an apparatus for packaging a substrate. The present apparatus includes a substrate having a first surface and a second surface. The first surface includes a probe array and the second surface is an outer periphery of the first surface. The present apparatus also includes a body having a mounting surface, an upper surface, and a cavity bounded by the mounting surface and the upper surface. The second surface is attached to the cavity and the first surface is within the cavity. A cover attached to the mounting surface for defining an upper boundary to the cavity is also included. The cavity includes a diffuser and a concentrator. The diffuser and the concentrator permit laminar fluid flow through the cavity.[0010]
• A further understanding of the nature and advantages of the inventions herein may be realized by reference to the remaining portions of the specification and the attached drawings.[0011]
BRIEF DESCRIPTION OF THE DRAWINGS
• FIG. 1[0012]aillustrates a wafer fabricated with a plurality of probe arrays.
• FIG. 1[0013]billustrates a chip.
• FIG. 2[0014]aillustrates a scribe and break device.
• FIG. 2[0015]billustrates the wafer mounted on a pick and place frame.
• FIGS. 2[0016]c-2dillustrate the wafer, as displayed by the scribe and break device during alignment.
• FIG. 3 illustrates a chip packaging device.[0017]
• FIG. 4 illustrates the chip packaging device assembled from two components.[0018]
• FIGS. 5[0019]a-5billustrate the top and bottom view of a top casing of the chip packaging device.
• FIG. 5[0020]cillustrates a different cavity orientation.
• FIG. 6 illustrates a cross sectional view of the packaging device.[0021]
• FIG. 7 illustrates the bottom view of a bottom casing of the chip packaging device.[0022]
• FIGS. 8[0023]a-8billustrate an acoustic welding system.
• FIGS. 9[0024]a-9cillustrate the acoustic welding process used in assembling the chip packaging device.
• FIG. 10 illustrates an adhesive dispensing system used in attaching the chip to the chip packaging device.[0025]
• FIGS.[0026]11-13 illustrate in greater detail the adhesive dispensing system of FIG. 10.
• FIGS. 14[0027]a-14dillustrate the procedure for aligning the system of FIG. 10.
• FIGS. 15[0028]a-15eillustrate images obtained during the alignment process of FIGS. 14a-14d.
• FIGS. 16[0029]a-16billustrate an alternative embodiment of a packaging device.
• FIGS. 17[0030]a-17billustrate another embodiment of a packaging device.
• FIG. 18 illustrates an alternative embodiment for attaching the chip to the packaging device.[0031]
• FIG. 19 illustrates another embodiment for attaching the chip to the packaging device.[0032]
• FIGS. 20[0033]a-20billustrate yet another embodiment for attaching the chip to the packaging device.
• FIG. 21 illustrates an alternative embodiment for attaching the chip to the packaging device.[0034]
• FIG. 22 illustrates another embodiment for attaching the chip to the packaging device.[0035]
• FIG. 23 illustrates an alternative embodiment for sealing the cavity on the packaging device.[0036]
• FIG. 24 illustrates another alternative embodiment for sealing the cavity on the packaging device.[0037]
• FIG. 25 illustrates yet another embodiment for sealing the cavity on the packaging device.[0038]
• FIGS. 26[0039]a-26billustrate an alternative embodiment for sealing the cavity on the packaging device.
• FIGS. 27[0040]a-27billustrate an alternative embodiment for mounting the chip.
• FIG. 28 illustrates an agitation system.[0041]
• FIG. 29 illustrates an alternative embodiment of the agitation system.[0042]
• FIG. 30 illustrates another embodiment of the agitation system.[0043]
• FIG. 31 illustrates an alternative embodiment of a chip packaging device.[0044]
• FIG. 32 illustrates side-views of the chip packaging device of FIG. 31.[0045]
• FIGS.[0046]33-35 illustrate in greater detail the chip packaging device of FIG. 31.
• FIG. 36 illustrates a further alternative embodiment of a chip packaging device.[0047]
DESCRIPTION OF THE SPECIFIC EMBODIMENTS CONTENTS
• I. Definitions[0048]
• II. General[0049]
• III. Details of One Embodiment of Invention[0050]
• a. Chip Package[0051]
• b. Assembly of Chip Package[0052]
• c. Chip Attachment[0053]
• IV. Details on Alternative Embodiments[0054]
• a. Chip Package[0055]
• b. Chip Attachment[0056]
• c. Fluid Retention[0057]
• d. Chip Orientation[0058]
• e. Parallel Diagnostics[0059]
• V. Details of an Agitation System[0060]
• I. Definitions[0061]
• The following terms are intended to have the following general meanings as they are used herein:[0062]
• 1. Probe: A probe is a surface-immobilized molecule that is recognized by a particular target and is sometimes referred to as a ligand. Examples of probes that can be investigated by this invention include, but are not restricted to, agonists and antagonists for cell membrane receptors, toxins and venoms, viral epitopes, hormones (e.g., opioid peptides, steroids, etc.), hormone receptors, peptides, enzymes, enzyme substrates, cofactors, drugs, lectins, sugars, oligonucleotides or nucleic acids, oligosaccharides, proteins, and monoclonal antibodies.[0063]
• 2. Target: A target is a molecule that has an affinity for a given probe and is sometimes referred to as a receptor. Targets may be naturally-occurring or manmade molecules. Also, they can be employed in their unaltered state or as aggregates with other species. Targets may be attached, covalently or noncovalently, to a binding member, either directly or via a specific binding substance. Examples of targets which can be employed by this invention include, but are not restricted to, antibodies, cell membrane receptors, monoclonal antibodies and antisera reactive with specific antigenic determinants (such as on viruses, cells or other materials), drugs, oligonucleotides or nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides, cells, cellular membranes, and organelles. Targets are sometimes referred to in the art as anti-probes or anti-ligands. As the term “targets” is used herein, no difference in meaning is intended. A “Probe Target Pair” is formed when two macromolecules have combined through molecular recognition to form a complex.[0064]
• II. General[0065]
• The present invention provides economical and efficient packaging devices for a substrate having an array of probes fabricated thereon. The probe arrays may be fabricated according to the pioneering techniques disclosed in U.S. Pat. No. 5,143,854 (Pirrung et al.), PCT WO 92/10092, or U.S. application Ser. No. 08/249,188 filed May 24, 1994, already incorporated herein by reference for all purposes. According to one aspect of the techniques described therein, a plurality of probe arrays are immobilized at known locations on a large substrate or wafer.[0066]
• FIG. 1[0067]aillustrates awafer100 on whichnumerous probe arrays110 are fabricated. Thewafer100 may be composed of a wide range of material, either biological, nonbiological, organic, inorganic, or a combination of any of these, existing as particles, strands, precipitates, gels, sheets, tubing, spheres, containers, capillaries, pads, slices, films, plates, slides, etc. The wafer may have any convenient shape, such as a disc, square, sphere, circle, etc. The wafer is preferably flat but may take on a variety of alternative surface configurations. For example, the wafer may contain raised or depressed regions on which a sample is located. The wafer and its surface preferably form a rigid support on which the sample can be formed. The wafer and its surface are also chosen to provide appropriate light-absorbing characteristics. For instance, the wafer may be a polymerized Langmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO2, SiN4, modified silicon, or any one of a wide variety of gels or polymers such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene, polycarbonate, or combinations thereof. Other materials with which the wafer can be composed of will be readily apparent to those skilled in the art upon review of this disclosure. In a preferred embodiment, the wafer is flat glass or single-crystal silicon.
• Surfaces on the solid wafer will usually, though not always, be composed of the same material as the wafer. Thus, the surface may be composed of any of a wide variety of materials, for example, polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, membranes, or any of the above-listed wafer materials.[0068]
• [0069]Wafer100 includes a plurality ofmarks145 that are located in streets150 (area adjacent to the probe arrays). Such marks may be used for aligning the masks during the probe fabrication process. In effect, the marks identify the location at which eacharray110 is to be fabricated. The probe arrays may be formed in any geometric shape. In some embodiments, the shape of the array may be squared to minimize wasted wafer area. After the probe arrays have been fabricated, the wafer is separated into smaller units known as chips. The wafer, for example, may be about 5×5 inches on which16 probe arrays, each occupying an area of about 12.8 cm², are fabricated.
• FIG. 1[0070]billustrates a chip that has been separated from the wafer. As illustrated,chip120 contains aprobe array110 and a plurality of alignment marks145. The marks serve multiple functions, such as: 1) aligning the masks for fabricating the probe arrays, 2) aligning the scriber for separating the wafer into chips, and 3) aligning the chip to the package during the attachment process. In some embodiments, such chips may be of the type known as Very Large Scale Immobilized Polymer Synthesis (VLSIPS™) chips.
• According to a specific embodiment, the chip contains an array of genetic probes, such as an array of diverse RNA or DNA probes. In some embodiments, the probe array will be designed to detect or study a genetic tendency, characteristic, or disease. For example, the probe array may be designed to detect or identify genetic diseases such as cystic fibrosis or certain cancers (such as P53 gene relevant to some cancers), as disclosed in U.S. patent application Ser. No. 08/143,312, already incorporated by reference.[0071]
• According to one embodiment, the wafer is separated into a plurality of chips using a technique known as scribe and break. FIG. 2[0072]aillustrates a fully programmable computer controlled scribe and break device, which in some embodiments is a DX-III Scriber breaker manufactured by Dynatex International™. As shown, thedevice200 includes a base205 with arotation stage220 on which a wafer is mounted. The rotation stage includes a vacuum chuck for fixing the wafer thereon. A stepper motor, which is controlled by the system, rotatesstage220. Located above the stage is ahead unit230 that includes acamera232 andcutter231.Head unit230 is mounted on a dual-axis frame. The camera generates an image of the wafer onvideo display210. Thevideo display210 includes a crosshair alignment mark215. The camera, which includes a zoom lens and a fiber optic light, allows a user to inspect the wafer on thevideo display210. A control panel240 is located on the base for operatingdevice200.
• In operation, a user places a[0073]wafer100 on aframe210 as illustrated in FIG. 2b. The surface offrame210 is composed of a flexible and sticky material. The tackiness of the frame prevents the chips from being dispersed and damaged during the breaking process.Frame210 may be a pick and place frame or a hoop that is commonly associated with fabrication of semiconductors. Referring back to FIG. 2a, a user places the frame with the wafer on therotation stage220. In some embodiments, the frame is held on the rotation stage by vacuum pressure. The user then aligns the wafer by examining the image displayed on thevideo display210.
• According to one embodiment, wafer alignment is achieved in two steps. First, using the control panel[0074]240, the user rotatesstage220. The stage is rotated untilstreets150 are aligned with thecross hair215 on the display, as illustrated in FIG. 2c. Next, the user moves the cutter until it is aligned at the center of one of the streets. This step is performed by aligninghorizontal line216 of the cross hair between alignment marks145, as shown in FIG. 2d.
• Once the cutter is aligned, the user instructs the device to scribe the wafer. In some embodiments, various options are available to the user, such as scribe angle, scribe pressure, and scribe depth. These parameters will vary depending on the composition and/or thickness of the wafer. Preferably, the parameters are set to scribe and break the wafer without causing any damage thereto or penetrating through the frame. The device repeatedly scribes the wafer until all the streets in one axis have been scribed, which in one embodiment is repeated 5 times (a 4×4 matrix of probe arrays). The user then rotates the stage 90° to scribe the perpendicular streets.[0075]
• Once the wafer has been scribed, the user instructs the device to break or separate the wafer into chips. Referring back to FIG. 2[0076]a, thedevice200 breaks the wafer by striking it beneath the scribe with an impulse bar located under the rotation table220. The shock from the impulse bar fractures the wafer along the scribe. Since most of the force is dissipated along the scribe,device200 is able to produce high breaking forces without exerting significant forces on the wafer. Thus, the chips are separated without causing any damage to the wafer. Once separated, the chips are then packaged. Of course, other more conventional techniques, such as the sawing technique disclosed in U.S. Pat. No. 4,016,855, incorporated herein by reference for all purposes, may be employed.
• III. Details of One Embodiment of the Invention[0077]
• a. Chip Package[0078]
• FIG. 3 illustrates a device for packaging the chips. Package[0079]300 contains acavity310 on which a chip is mounted. The package includesinlets350 and360 which communicate withcavity310. Fluids are circulated through the cavity viainlets350 and360. A septum, plug, or other seal may be employed to seal the fluids in the cavity. Alignment holes330 and335 may be provided for alignment purposes. In some embodiments, the package may include anon-flush edge320. In some detection systems, the packages may be inserted into a holder similar to an audio cassette tape. The asymmetrical design of the package will assure correct package orientation when inserted into the holder.
• FIG. 4 illustrates one embodiment of the package. As shown in FIG. 4, the chip package is manufactured by mating two substantially[0080]complementary casings410 and420 to formfinished assembly300. Preferably,casings410 and420 are made from injection molded plastic. Injection molding enables the casings to be formed inexpensively. Also, assembling the package from two parts simplifies the construction of various features, such as the internal channels for introducing fluids into the cavity. As a result, the packages may be manufactured at a relatively low cost.
• FIGS. 5[0081]a-5bshow thetop casing410 in greater detail. FIG. 5ashows a top view and FIG. 5bshows a bottom view. Referring to FIG. 5a,top casing410 includes an externalplanar surface501 having acavity310 therein. In some embodiments, the surface area ofcasing410 sufficiently accommodates the cavity. Preferably, the top casing is of sufficient size to accommodate identification labels or bar codes in addition to the cavity. In a specific embodiment, the top casing is about 1.5″ wide, 2″ long, and 0.2″ high.
• [0082]Cavity310 is usually, though not always, located substantially at the center ofsurface501. The cavity may have any conceivable size, shape, or orientation. Preferably, the cavity is slightly smaller than the surface area of the chip to be placed thereon and has a volume sufficient to perform hybridization. In one embodiment, the cavity may be about 0.58″ wide, 0.58″ long, and 0.2″ deep.
• [0083]Cavity310 may includeinlets350 and360. Selected fluids are introduced into and out of the cavity via the inlets. In some embodiments, the inlets are located at opposite ends of the cavity. This configuration improves fluid circulation and regulation of bubble formation in the cavity. The bubbles agitate the fluid, increasing the hybridization rate between the targets and complementary probe sequences. In one embodiment, the inlets are located at the top and bottom end of the cavity when the package is oriented vertically such as at the opposite corners of the cavity. Locating the inlet at the highest and lowest positions in the cavity facilitates the removal of bubbles from the cavity.
• FIG. 5[0084]cillustrates an alternative embodiment in whichcavity310 is oriented such that the edges of thecavity310 and thecasing410 are non-parallel. This configuration allowsinlets350 and360 to be situated at the absolute highest and lowest locations in the cavity when the package is vertically oriented. As a result, bubbles or fluid droplets are prevented from being potentially trapped in the cavity.
• Referring back to FIG. 5[0085]a, adepression550 surrounds the cavity. In some embodiments, aridge560 may be provided at the edge of the depression so as to form a trough. The ridge serves to support the chip above the cavity. To attach the chip to the package, an adhesive may be deposited in the trough. This configuration promotes efficient use of chip surface area, thus increasing the number of chips yielded from a wafer.
• [0086]Top casing410 includes alignment holes330 and335. In some embodiments, holes330 and335 are different in size to ensure correct orientation of the package when mounted on an alignment table. Alternatively, the holes may have different shapes to achieve this objective. Optionally, the holes taper radially inward fromsurface501 toward502 to reduce the friction against alignment pins while still maintaining adequate contact to prevent slippage.
• Referring to FIG. 5[0087]b,channels551 and561 are optionally formed oninternal surface502.Channels551 and561 communicate withinlets350 and360 respectively. Adepression590 is formed below cavity. According to some embodiments, the shape ofdepression590 is symmetrical to the cavity with exception tocorners595 and596, which accommodate the inlets. The depth ofdepression590 may be, for example, about 0.7″. As a result, the bottom wall of the cavity is about 0.05″ thick.Depression590 may receive a temperature controller to monitor and maintain the cavity at the desired temperature. By separating the temperature controller and cavity with a minimum amount of material, the temperature within the cavity may be controlled more efficiently and accurately. Alternatively, channels may be formed onsurface502 for circulating air or water to control the temperature within the cavity.
• In some embodiments,[0088]certain portions595 ofinternal surface502 may be eliminated or cored without interfering with the structural integrity of the package when assembled. Coring the casing reduces the wall thickness, causing less heat to be retained during the injection molding process; potential shrinkage or warpage of the casing is significantly reduced. Also, coring decreases the time required to cool the casing during the manufacturing process. Thus, manufacturing efficiency is improved.
• In one embodiment, the top casing and bottom casing are mated together using a technique known as acoustic or ultrasonic welding. Accordingly, “energy directors”[0089]510 are provided. Energy directors are raised ridges or points, preferably v-shaped, that are used in an acoustic welding process. The energy directors are strategically located, for example, to seal the channels without interfering with other features of the package and to provide an adequate bond between the two casings. Alternatively, the casings may be mated together by screws, glue, clips, or other mating techniques.
• FIGS.[0090]6 shows a cross sectional view of thecavity310 withchip120 mounted thereon in detail. As shown, adepression550 is formed aroundcavity310. The depression includes aridge560 which supportschip120. The ridge and the depression create a trough aroundcavity310. In some embodiments, the trough is sufficiently large to receive an adhesive630 for attaching the chip to the package. In one embodiment, the trough is about 0.08″ wide and 0.06″ deep. When mounted, the edge of the chip protrudes slightly beyondridge550, but without contactingside625 of the depression. This configuration permits the adhesive to be dispensed onto the trough and provides adequate surface area for the adhesive to attachchip120 to the package.
• According to some embodiments, the[0091]back surface130 ofchip120 is at least flush or below the plane formed bysurface501 ofcasing410. As a result,chip120 is shielded bysurface501 from potential damage. This configuration also allows the packages to be easily stored with minimal storage area since the surfaces are substantially flat.
• Optionally, the bottom of the cavity includes a light absorptive material, such as a glass filter or carbon dye, to prevent impinging light from being scattered or reflected during imaging by detection systems. This feature improves the signal-to-noise ratio of such systems by significantly reducing the potential imaging of undesired reflected light.[0092]
• FIG. 7 shows the internal surface of[0093]bottom casing420 in greater detail. As shown, thebottom casing420 is substantially planar and contains anopening760 therein. Preferably, thecasing420 is slightly wider or slightly longer than the top casing. In one embodiment, casing420 is about 1.6″ wide, 2.0″ long, and 0.1″ deep, which creates a non-flush edge on the finish assembly. As previously mentioned, this design ensures that the package is correctly oriented when mounted onto the detection systems.
• In some embodiments, opening[0094]760 is spatially located at about the depression below the cavity. The opening also has substantially the same geometric configuration as the depression to allow the temperature controller to contact as much of the bottom of the cavity as possible.
• [0095]Internal surface701 ofcasing420 includesdepressions730 and740. Aport731 is located indepression730 and aport741 is located indepression740.Ports731 and741 communicate with channels on the top casing (350 and360 in FIG. 5b) when the package is assembled. Aseal790, which may be a septum composed of rubber, teflon/rubber laminate, or other sealing material is provided for each depression. The septum may be of the type commonly used to seal and reseal vessels when a needle is inserted into the septum for addition/removal of fluids. The septums, when seated in the depressions, extend slightly above surface, which in some embodiments is about 0.01″.
• This design causes[0096]casings410 and420 to exert pressure on the septum, forming a seal between the ports and the channels. The seal is maintained even after fluid is injected into the cavity since the pressure immediately forces the septum to reseal itself after the needle or other fluid injecting means is removed from the port. Thus, an efficient and economical seal for retaining fluid in the cavity is provided.
• Also, casing[0097]420 includes the complementaryhalf alignment holes330 and335, each tapering radially inward from the external surface. Further,certain areas765 oninternal surface701 may be cored, as similar to the internal surface of the top casing.
• FIG. 31 is a simplified illustration of an alternative embodiment of a[0098]chip packaging device3100 according to the present invention. The chip packaging device includes a plurality ofcasings3200,3300, and3400. The casings may be defined as atop casing3200, amiddle casing3300, and abottom casing3400. The casings are made of known plastic materials such as ABS plastic, polyvinylchloride, polyethylene, products sold under the trademarks TEFLON™ and KALREZ™ and the like, among others. Preferably, the casings can be made by way of injection molding and the like. Assembling the chip packaging device from three casings simplifies construction for the fabrication of internal channels and the like, and can also be made at a relatively low cost.
• Support structures (or alignment holes) exist at selected locations of the chip packing device. The support structures can be used to mount or position the chip packaging device to an apparatus, e.g., scanner or the like. In an embodiment, the[0099]top casing3200 includessupport structures3201 and3203 on each side of acenter opening3209. Themiddle casing3300 includessimilar support structures3313 and3315 which are complementary to thesupport structures3201 and3203, respectively, in the top casing. The bottom casing also includessimilar support structures3403 and3401, respectively, which are complementary to the support structures in the top casing and the middle casing. As shown, each of the support structures on each side of the center opening align with each other. Each support structure is, for example, an aperture through the casing. The aperture includes an outer periphery defined by a geometrical shape which may be round, rectangular, trapezoidal, hexagonal, or the like.
• The present chip packaging device assembles with use of complementary alignment pins and bores on the casings. By way of alignment pins (not shown), the top casing aligns with and inserts into alignment bores[0100]3301,3303 in themiddle casing3300. Alternatively, the middle casing can have alignment pins or the like and the top casing has the alignment bores or the like. The bottom casing includesalignment pins3407 and3409 which align to and insert into alignment bores (not shown) in bottom portions of the middle casing. The use of alignment bores and pins provide for ease in assembly of the chip carrier. Upon assembly, the alignment bores and pins on the casings prevent the casings from moving laterally relative to each other.
• A[0101]center opening3209 in the top casing overlies acenter portion3317 of themiddle casing3300. Thecenter portion3317 of the middle casing includes an inner annular region (or cavity edges) with a bottom portion which is preferably a flat bottom portion. The flat bottom portion of the middle casing and portions of the bottom casing including edges define acavity3405. A chip is placed overlying an underlying portion of thecavity3407.
• Optionally, a temperature control mechanism such as a heater, a cooler, or a combination thereof is disposed into the center opening against the bottom portion of the middle casing. The temperature control mechanism can be any suitable thermally controlled element such as a resistive element, a temperature controlled block or mass, thermoelectric modules, or the like. The temperature control mechanism transfers heat via conduction to the bottom center portion, which transfers heat to, for example, fluid in the cavity or the chip. Alternatively, the temperature control mechanism sinks heat away from, for example, fluid in the cavity or the chip through the bottom center portion. The temperature control mechanism maintains a selected temperature in the cavity. The temperature control mechanism also includes a temperature detection device such as a thermocouple which provides signals corresponding to temperature readings. A controller receives the signals corresponding to the temperature readings, and adjusts power output to the temperature control mechanism to maintain the selected temperature.[0102]
• The[0103]top casing3200 also includeschannels3205 and3207 for fluid transfer. Thechannels3205 and3207 communicate withannular regions3309 and3311, respectively, on themiddle casing3300 for fluid transfer. A septum, a plug, an o-ring, a gasket, or the like viaannular regions3309 and3311 seals fluids within thetop casing channels3205 and3207 and the middle casing. The bottom casing includeschannels3411 and3413 in communication withchannels3307 and3305, respectively. A septum, a plug, an o-ring, a gasket, or the like seals the fluids within thebottom casing channels3411 and3413 and themiddle casing channels3305 and3307.
• The chip packaging device provides an even distribution of fluid (or fluid flow) through the cavity over a top surface (or inner or active surface) of the chip. For example, a selected fluid enters[0104]channel3207, flows throughchannel3307, changes direction and flows throughchannel3411, and evenly distributes into thecavity3405 over the top surface of the chip. As previously noted, the cavity is defined by the flat bottom portion and cavity edges. A selected fluid exits the cavity by way ofchannel3413,channel3305, andchannel3205. The fluid flow over the top surface of the chip is preferably laminar, but may also be turbulent, a combination thereof or the like. By way of the present chip packaging device, a substantial portion of turbulent flow remains at an upper portion of thechannel3411, and does not enter the cavity.
• Preferably, a selected fluid enters the cavity by way of[0105]channel3205,channel3305, andchannel3413. The selected fluid exits the cavity throughchannel3411,channel3307, andchannel3207. In a preferred embodiment, the fluid flows against the direction of gravity through the cavity. Of course, other fluid flow routes may also be employed depending upon the particular application.
• FIG. 32 illustrates an assembled[0106]chip packaging device3100 according to the present invention. As shown are a top-view3200, a side-view3500, a bottom-view3400, and a front-view3600 of the assembledchip packaging device3100. The assembledchip packaging device3100 includes thebottom casing3400, themiddle casing3300, and thetop casing3200.
• The top-[0107]view3200 of the top casing includesalignment structures3205,3215 surroundingopening3209. Theopening3209 includes a bevelledannular region3211 surrounding the periphery of thechannel3209. The alignment bores3203 and3201 also include bevelledannular regions3213 and3215, respectively. A bevelledannular region3217,3221 also surrounds eachfluid channel3205,3207 to assist with fluid flow therethrough.
• The bottom-[0108]view3400 of the bottom casing includesalignment structures3401,3403 surrounding thecavity3405. The cavity includes a flat bottomperipheral portion3415, abevelled portion3417 extending from the flat bottom peripheral portion, and a flatupper portion3419 surrounding the bevelled portion. The chip includes an outer periphery which rests against the flat bottomperipheral portion3415. The bevelled portion aligns the chip onto the flat bottomperipheral portion3415. Similar to the previous embodiments, the top casing extends outside3421 the middle and bottom casings.
• The[0109]cavity3405 is preferably located at a center of the bottom casing, but may also be at other locations. The cavity may be round, square, rectangular, or any other shape, and orientation. The cavity is preferably smaller than the surface area of the chip to be placed thereon, and has a volume sufficient to perform hybridization and the like. In one embodiment, the cavity includes dimensions such as a length of about 0.6 inch, a width of about 0.6 inch and a depth of about 0.07 inch.
• In a preferred embodiment, the bottom casing with selected cavity dimensions may be removed from the middle and top casings, and replaced with another bottom casing with different cavity dimensions. This allows a user to attach a chip having a different size or shape by changing the bottom casing, thereby providing ease in using different chip sizes, shapes, and the like. Of course, the size, shape, and orientation of the cavity will depend upon the particular application.[0110]
• FIGS.[0111]33-35 illustrate in greater detail the chip packaging device of FIG. 31. FIG. 33 illustrates simplified top-view3260 and bottom-view3250 diagrams of thetop casing3200. As shown, the reference numerals refer to the same elements as the top casing of FIG. 31. FIG. 34 illustrates a simplified top-view3350 and bottom-view3360 diagrams of themiddle casing3300. As shown, the reference numerals refer to the same elements as the middle casing of FIG. 31. In addition, the bottom-view of the casing includes a substantially smooth andplanar bottom surface3361. A portion of the bottom surface defines an upper portion of the cavity. But the bottom surface can also be textured, ridged, or the like to create turbulence or a selected fluid flow through the cavity. The bottom surface is preferably a hydrophobic surface which enhances laminar flow through the cavity. Of course, the type of bottom surface depends upon the particular application.
• FIG. 35 illustrates simplified top-[0112]view3460 and bottom-view3450 diagrams of thebottom casing3400. As shown, the reference numerals refer to the same elements as the bottom casing of FIG. 31. In an embodiment, fluid fromchannel3305 changes direction at anupper portion3431 of the channel and flows to alower portion3433 of the channel. Fluid evenly distributes from thelower portion3433 via afluid distribution point3435. The distributed fluid evenly passes over a slanted edge (or bevelled edge)3437 which drops fluid evenly to a top surface of the chip in the cavity. By way of slantededge3427 which slopes up to afluid concentration point3425, fluid leaves the cavity and enters thechannel3411. In particular, fluid leaves the cavity and enters alower portion3423 of the channel, flows through the channel, and changes directions at anupper portion3421 of the channel. Each channel includes a length L and a width W. The distribution point and the concentration point are positioned at a distance away from the cavity to substantially prevent turbulence from forming in the cavity, and in particular over the top surface of the chip. The channels are each angled at an angle Θ ranging from about 2 degrees to about 90 degrees, but is preferably about 5 degrees to about 45 degrees. The angle enhances an even distribution of laminar flow into the cavity. Of course, the exact angle, channel shape, and dimensions depend upon the particular application.
• FIG. 36 illustrates a simplified cross-sectional view of an[0113]alternative embodiment3600 of the chip packaging device. The chip packaging device includes the threecasings3200,3300, and3400 of the previous embodiment, and also includes hollow pins, needles, or the like3601 and3603. Each of the pins transfers a selected fluid to and from thecavity3405. Preferably, eachpin3601 includes anexternal opening3609, atubular region3611, aninner opening3607, apointed tip3605, and other elements. The pin is made from a suitable material such as a glass, a stainless steel or any other high quality material to transfer fluids to and from thecavity3405.
• In a preferred embodiment, each pin is inserted into its[0114]channel region3205 or3207. A point on the pin tip pierces through, for example, a septum at anannular region3309 or3311. A selected fluid travels through pin3603 (throughchannel3205 and at least a portion of3305), enters the upper region ofchannel3413, and into thecavity3405. The selected fluid travels from the cavity, throughpin3601, and to the external apparatus. Alternatively, the selected fluid enters the cavity viapin3601 and exits the cavity viapin3603. The selected fluid may also enter the cavity via pin and exit the cavity through the channels without use of a pin. The selected fluid may further enter the cavity through the channels without use of a pin and exit through a pin. Of course, the particular pin used and fluid flow will depend upon the application.
• It should be noted that the even distribution of fluid flow through the cavity prevents “hot spots” from occurring in the cavity. For example, the even distribution of fluid through the cavity by way of the previous embodiment substantially prevents fluid from becoming substantially turbulent at certain locations. This prevents “hot spots” caused by such turbulent fluid. The hot spots are often caused by higher chemical activity or exothermic reactions and the like by way of turbulence in such certain locations.[0115]
• b. Assembly of Chip Package[0116]
• According to one embodiment, the top and bottom casing are attached by a technique known as ultrasonic or acoustic welding. FIG. 8[0117]ais a schematic diagram of acoustic welding system used for assembling the package. In some embodiments, thewelding system800 is a HS Dialog ultrasonic welder manufactured by Herrmann Ultrasonics Inc.System800 includes aplatform850 mounted onbase810.Platform850 accommodates the top and bottom casings during the assembling process.
• An[0118]acoustic horn860 is mounted on a frame aboveplatform850. The horn translates vertically (toward and away from platform850) on the frame by air pressure. The horn is connected to afrequency generator870, which in some embodiments is a 20 KHz generator manufactured by Herrmann Ultrasonics Inc.System800 is controlled by acontroller880, which, for example, may be a Dialog 2012 manufactured by Herrmann Ultrasonics Inc.Controller880 may be configured to accept commands from adigital computer system890.Computer890 may be any appropriately programmed digital computer of the type that is well known to those skilled in the art such as a Gateway 486DX operating at 33 MHz.
• FIG. 8[0119]billustratesplatform850 in greater detail. Theplatform850 is substantially planar and includes alignment pins851 and852. Alignment pins851 and852 are used to align both the top and bottom casings during the welding process. In some embodiments, apad890, which may be composed of silicone rubber or other energy absorbing material, is located onplatform850 to prevent damage to the package during assembly.
• FIG. 9[0120]aillustrates the acoustic welding system in operation. As shown,bottom casing420, having aseptum790 seated in each depression, is mounted onto platform table850 and held in place by alignment pins.Top casing410 is then aligned above the bottom casing with alignment pins. The system then commences the welding process by loweringhorn860 until it contacts the top surface ofcasing410.
• FIG. 9[0121]billustrates the casing and horn in detail. As shown, thehorn860 presses againsttop casing410, thereby forcingenergy directors510 to interface withbottom casing420. The system then activates the frequency generator, causing the welding horn to vibrate.
• FIG. 9[0122]cillustrates in detail the energy directors during the welding process. As shown instep9001, weldinghorn860forces energy directors510 againstbottom casing420. Atstep9002, the system vibrates the welding horn, which in some embodiments is at 20 KHz. The energy generated by the horn melts the energy directors. Simultaneously, the horn translates downward against the package. Atstep9003, the pressure exerted by the horn causes the energy directors to fuse with the bottom casing. Atstep9004, the welding process is completed when the horn reaches its weld depth, for example, of about 0.01″. Of course, the various welding parameters may be varied, according to the composition of the materials used, to achieve optimum results.
• c. Chip Attachment[0123]
• According to some embodiments, an ultraviolet cured adhesive attaches the chip to the package. FIG. 10 schematically illustrates an adhesive dispensing system used in attaching the chip. The[0124]dispensing system1000 includes an attachment table1040 to accommodate the package during the attachment process. A chip alignment table1050 for aligning the chip is located adjacent to attachment table1040. Ahead unit1030 for dispensing the adhesive is located above tables1040 and1050. Thehead unit1030 also includes a camera that generates an output tovideo display1070.Video display1070, in some embodiments, includes a crosshair alignment mark1071. The head unit is mounted on a dual-axis (x-y) frame for positioning during alignment and attachment of the chip. The operation of the dispensing system is controlled by acomputer1060, which in some embodiments may be Gateway 486DX operating at 33 MHz.
• FIG. 11 illustrates the attachment table in greater detail. The attachment table[0125]1040 has a substantiallyflat platform1110 supported by a plurality oflegs1105. Alignment pins1115 and1116, which secure the package during the attachment process, are located on the surface ofplatform1110.
• Optionally, a[0126]needle1120 is provided.Needle1120 includes achannel1121 and is connected to a vacuum pump. In operation, the needle is inserted into one of the ports of the package in order to generate a vacuum in the cavity. The vacuum pressure secures the chip to the package during the attachment process.
• FIG. 12[0127]ashows table1050 in greater detail. Table1050 includes a substantiallyflat platform1210 having adepression1240 for holding a chip. In some embodiments, aport1241 is provided indepression1240.Port1241 is connected to a vacuum pump which creates a vacuum in the depression for immobilizing the chip therein.Platform1210 is mounted on a combination linearrotary stage1246, which in some embodiments may be a model 26LR manufactured by DARDAL, and a singleaxis translation stage1245, which may be a model CR2226HSE2 manufactured by DARDAL.
• FIG. 12[0128]billustratesdepression1240 in greater detail. As shown, aledge1241 surrounds thedepression1240.Ledge1241 supports the chip when it is placed abovedepression1240. Since the chips are placed over the depression with the probes facing the table, this design protects the probes from being potentially damaged during alignment.
• FIG. 13 illustrates the[0129]head unit1030 in greater detail. As shown, thehead unit1030 includes acamera assembly1320 that generates an output to a video display. A light1360 is provided to enable the camera to focus and image an object of interest. The head unit also includes anultraviolet light1350 for curing the adhesive, avacuum pickup1330 for moving chip during the attachment process, and anadhesive dispenser1340.
• In operation, a chip package is placed onto table[0130]1040. As previously described, the alignment pins on the table immobilize the package. The user begins the chip attachment process by calibrating the head unit. This may be done by moving the camera above the package and aligning it with a mark on the package, as shown in FIG. 14a. For convenience, one of the alignment pins may be used as an alignment mark. FIG. 14billustrates atypical image1440 generated by the camera during this step. As shown, the head unit is not aligned withpin1480. To align the head unit, the user translates it in both the x and y direction untilpin1480 is located at theintersection1477 of the cross hair on the video display, as illustrated in FIG. 14c.
• Next, the chip is inserted into the depression on the chip alignment table. FIG. 14[0131]cis a flow chart indicating the steps for aligning the chip. Atstep1410, the system positions the camera (head unit) above one of the chip's alignment marks. The camera images the alignment mark on the video display. At this point, the mark is normally misaligned (i.e., the mark is not located at the intersection of the cross hair alignment mark). Atstep1420, the user adjusts the chip alignment table in both the x and y direction until the mark is substantially located at the intersection of the cross hair. Since no rotational adjustments were made, the mark may be misaligned angularly.
• At[0132]step1430, the user instructs the system to move the camera above a second alignment mark, which usually is at an opposite corner of the chip. Again, an image of the alignment mark is displayed. At this stage, the alignment mark is probably misaligned in the x, y, and angular directions. Atstep1440, the user adjusts the rotational stage, x-stage, and y-stage, if necessary, to align the mark with the cross hair on the video display. In instances where the rotational stage has been rotated, the first alignment mark will become slightly misaligned. To compensate for this shift, the user repeats the alignment process beginning atstep1450 until both marks are aligned. Of course, image processing techniques may be applied for automated head unit and chip alignment.
• FIG. 15[0133]ais an example of an image displayed by the video screen duringstep1410. As shown, the first alignment mark (lower left corner of the chip) is not aligned with the cross hair marking. FIG. 15bexemplifies an image of the first alignment mark after adjustments were made by the user. FIG. 15cillustrates a typical image displayed by video screen duringstep1430. As illustrated, the second alignment mark (upper right corner of the chip) is misaligned in the x, y, and angular directions. FIG. 15dillustrates an image of the second mark following initial adjustments by the user atstep1440. FIG. 15eillustrates the orientation of the second alignment mark after the chip has been aligned.
• Once the chip is aligned, the vacuum holding the chip on the attachment table is released. Thereafter, the pickup on the head unit removes the chip from the table and aligns it on the cavity of the package. In some embodiments, the chip is mated to the pickup by a vacuum.[0134]
• Optionally, the user may check to ensure that the chip is correctly aligned on the cavity by examining the chip's alignment marks with the camera. If the chip is out of position, the chip is removed and realigned on the alignment table. If the chip is correctly positioned, the system deposits an adhesive by moving the dispenser along the trough surrounding the cavity. In some embodiments, the vacuum is released before depositing the adhesive in the trough. This step is merely precautionary and implemented to ensure that the vacuum does not cause any adhesive to seep into the cavity. Once the adhesive is deposited, the system reexamines the chip to determine if the adhesive had moved the chip out of position. If the chip is still aligned, the head unit locates the ultraviolet light above the adhesive and cures it for a time sufficient to harden the adhesive, which in one embodiment is about 10 seconds. Otherwise, the chip is realigned.[0135]
• Upon completion, the chip package will have a variety of uses. For example, the chip package will be useful in sequencing genetic material by hybridization. In sequencing by hybridization, the chip package is mounted on a hybridization station where it is connected to a fluid delivery system. Such system is connected to the package by inserting needles into the ports and puncturing the septums therein. In this manner, various fluids are introduced into the cavity for contacting the probes during the hybridization process.[0136]
• Usually, hybridization is performed by first exposing the sample with a prehybridization solution. Next, the sample is incubated under binding conditions with a solution containing targets for a suitable binding period. Binding conditions will vary depending on the application and are selected in accordance with the general binding methods known including those referred to in: Maniatis et al.,[0137]Molecular Cloning: A Laboratory Manual(1989), 2nd Ed., Cold Spring Harbor, N.Y. and Berger and Kimmel,Methods in Enzymology, Volume152, Guide to Molecular Cloning Techniques(1987), Academic Press, Inc., San Diego, Calif.; Young and Davis (1983)Proc. Natl. Acad. Sci. (U.S.A.) 80: 1194, which are incorporated herein by reference. In some embodiments, the solution may contain about 1 molar of salt and about 1 to 50 nanomolar of targets. Optionally, the fluid delivery system includes an agitator to improve mixing in the cavity, which shortens the incubation period. Finally, the sample is washed with a buffer, which may be 6×SSPE buffer, to remove the unbound targets. In some embodiments, the cavity is filled with the buffer after washing the sample.
• Thereafter, the package may be aligned on a detection or imaging system, such as those disclosed in U.S. Pat. No. 5,143,854 (Pirrung et al.) or U.S. patent application Ser. No. 08/495,889 (Attorney Docket Number 11509-117), already incorporated herein by reference for all purposes. Such detection systems may take advantage of the package's asymmetry (i.e., non-flush edge) by employing a holder to match the shape of the package specifically. Thus, the package is assured of being properly oriented and aligned for scanning. The imaging systems are capable of qualitatively analyzing the reaction between the probes and targets. Based on this analysis, sequence information of the targets is extracted.[0138]
• IV. Details on Alternative Embodiments[0139]
• a. Chip Package Orientation[0140]
• FIGS. 16[0141]a-16billustrate an alternative embodiment of the package. FIG. 16ashows a top view and FIG. 16bshows a bottom view. As shown in FIG. 16a, acavity1620 is located on atop surface1610 of thepackage body1600. The body includesalignment holes1621 and1622 that are used, for example, in mating the chip to the package. Optionally, a plurality ofridges1690 is located atend1660 of the body. The friction created byridges1690 allows the package to be handled easily without slippage.
• The body also includes two substantially[0142]parallel edges1630 and1640. As shown,edge1640 is narrowed atend1665 to create anuneven edge1645. The asymmetrical design of the body facilitates correct orientation when mounted onto detection systems. For example, detection systems may contain a holder, similar to that of an audio cassette tape, in which end1665 is inserted.
• Referring to FIG. 16[0143]b,ports1670 and1671 communicate withcavity1620. A seal is provided for each port to retain fluids in the cavity. Similar to the top surface, the bottom surface may optionally include a plurality ofridges1690 atend1660.
• FIGS. 17[0144]a-17billustrate an alternative embodiment of the package. FIG. 17ashows a top view and FIG. 17bshows a bottom view. Referring to FIG. 17a, acavity1720 is located on atop surface1710 of thepackage body1700. The body may be formed in the shape of a disk with two substantiallyparallel edges1730 and1740. Alignment holes1721 and1722, which may be different in size or shape, are located on the body. In some embodiments, the package is inserted like an audio cassette tape into detection systems in a direction parallel toedges1730 and1740.Edges1730 and1740 and alignment holes prevent the package from being inserted incorrectly into the detection systems.
• As shown in FIG. 17[0145]b,ports1730 and1740 are located on thebottom surface1715 of the package.Ports1730 and1740 communicate withcavity1720 and each include aseal1780 for sealing fluids in the cavity.
• b. Chip Attachment[0146]
• FIG. 18 illustrates an alternative embodiment for attaching the chip to the package. As shown, two[0147]concentric ledges1810 and1820 surround the perimeter ofcavity310.Ledge1820 supports thechip120 when mounted abovecavity310.Ledge1810, which extends beyondchip120, receives an adhesive1860 such as ultraviolet cured silicone, cement, or other adhesive for attaching the chip thereto.
• FIG. 19 illustrates another embodiment for attaching the chip to the package. According to this embodiment, a[0148]ledge1910 is formed aroundcavity310. Preferably, the ledge is sufficiently large to accommodate an adhesive1920 such as an adhesive film, adhesive layer, tape, or any other adhesive layer.Chip120 attaches to the package when it contacts the adhesive film.
• FIG. 20[0149]aillustrates yet another embodiment for attaching a chip to the package. As shown, aclamp2010, such as a frame having a plurality offingers2015, attaches the chip to the package. FIG. 20billustrates a cross sectional view. Aridge2020 onsurface501 surroundscavity310. The ridge includes aledge2025 upon whichchip120 rests. Optionally, a gasket or aseal2070 is located between the ledge and chip to ensure a tight seal aroundcavity310.Clamp2010 is attached toside2040 ofridge2020 andsurface501. In some embodiments,clamp2010 is acoustically welded to the body. Accordingly,clamp2010 includesenergy directors2050 located at its bottom. Alternatively, screws, clips, adhesives, or other attachment techniques may be used to mateclamp2010 to the package. When mated,fingers2015secure chip120 to the package.
• FIG. 21 illustrates an alternative embodiment for attaching the chip to the package. A[0150]ridge2110, having anotch2115 at or near the top ofridge2110, encompasses thecavity310.Chip120 is wedged and held into position bynotch2115. Thereafter, a process known as heat staking is used to mount the chip. Heat staking includes applying heat and force atside2111 of ridge, thus forcing ridge tightly against or aroundchip120.
• FIG. 22 shows another embodiment of attaching a chip onto a package. As shown, a[0151]channel2250 surroundscavity310. Anotch2240 for receiving thechip120 is formed along or near the top of thecavity310. In some embodiments, a gasket orseal2270 is placed at the bottom of the notch to ensure a tight seal when the chip is attached. Once the chip is located at the notch, a V-shapedwedge2260 is inserted intochannel2250. The wedge forces the body to press against chip's edges andseal2260, thus mating the chip to the package. This process is known as compression sealing.
• Other techniques such as insert molding, wave soldering, surface diffusion, laser welding, shrink wrap, o-ring seal, surface etching, or heat staking from the top may also be employed.[0152]
• c. Fluid Retention[0153]
• FIG. 23 shows an alternative embodiment of package that employs check valves to seal the inlets. As shown,[0154]depressions2305 and2315 communicate withcavity310 throughinlets350 and360. Checkvalves2310 and2320, which in some embodiments may be duck-billed check valves, are seated indepressions2305 and2315. To introduce a fluid into the cavity, a needle is inserted into the check valve. When the needle is removed, the check valve reseals itself to prevent leakage of the fluid.
• FIG. 24 illustrates another package that uses reusable tape for sealing the[0155]cavity310. As shown, atape2400 is located aboveinlets350 and360. Preferably,end2430 of tape is permanently fixed tosurface2480 whileend2410 remains unattached. Themid section2420 of the tape is comprised of non-permanent adhesive. This design allows inlets to be conveniently sealed or unsealed without completely separating the tape from the package.
• FIG. 25 illustrates yet another embodiment of the package that uses plugs to retain fluids within the cavity. As shown,[0156]depressions2520 and2530 communicate withcavity310 viainlets350 and360. Aplug2510, which in some embodiment may be composed of rubber or other sealing material, is mated to each of the depressions.Plugs2510 are easily inserted or removed for sealing and unsealing the cavity during the hybridization process.
• FIG. 26[0157]aillustrates a package utilizing sliding seals for retaining fluids within the cavity. The seals are positioned inslots2610 that are located above the inlets. The slots act as runners for guiding the seals to and from the inlets. FIG. 26billustrates the seal in greater detail.Seal2640, which may be composed of rubber, teflon rubber, or other sealing material, is mated to eachslot2610. The seal includes ahandle2650 which extends through the slot. Optionally, the bottom of the seal includes anannular protrusion2645 to ensure mating withinlet350. The inlet is sealed or unsealed by positioning the seal appropriately along the slot. Alternatively, spring loaded balls, rotary ball valves, plug valves, or other fluid retention techniques may be employed.
• d. Chip Orientation[0158]
• FIGS. 27[0159]a-27billustrate an alternative embodiment of the package. FIG. 27aillustrates a top view and FIG. 27bshows a cross sectional view. As shown,package2700 includes acavity2710 on asurface2705. Achip2790 having an array ofprobes2795 onsurface2791 is mated to the bottom ofcavity2710 with an adhesive2741. The adhesive, for example, may be silicone, adhesive tape, or other adhesive. Alternatively, clips or other mounting techniques may be employed. Optionally, the bottom of the cavity may include a depression in which a chip is seated.
• This configuration provides several advantages such as: 1) permitting the use of any type of substrate (i.e., non-transparent or non-translucent), 2) yielding more chips per wafer since the chip does not require an edge for mounting, and 3) allowing chips of various sizes or multiple chips to be mated to the package.[0160]
• A[0161]cover2770 is mated to the package for sealing the cavity. Preferably,cover2770 is composed of a transparent or translucent material such as glass, acrylic, or other material that is penetrable by light.Cover2270 may be mated to surface2705 with an adhesive2772, which in some embodiments may be silicone, adhesive film, or other adhesive. Optionally, a depression may be formed around the cavity such that surface2271 of the cover is at least flush withsurface2705. Alternatively, the cover may be mated to surface2705 according to any of the chip attachment techniques described herein.
• [0162]Inlets2750 and2751 are provided and communicate withcavity2710. Selected fluids are circulated through the cavity viainlets2750 and2751. To seal the fluids in the cavity, a septum, plug, or other seal may be employed. In alternative embodiments, any of the fluid retention techniques described herein may be utilized.
• e. Parallel Hybridization and Diagnostics[0163]
• In an alternative embodiment, the body is configured with a plurality of cavities. The cavities, for example, may be in a[0164]96-well micro-titre format. In some embodiments, a chip is mounted individually to each cavity according to the methods described above. Alternatively, the probe arrays may be formed on the wafer in a format matching that of the cavities. Accordingly, separating the wafer is not necessary before attaching the probe arrays to the package. This format provides significant increased throughput by enabling parallel testing of a plurality of samples.
• V. Details of an Agitation System[0165]
• FIG. 28 illustrates an agitation system in detail. As shown, the[0166]agitation system2800 includes twoliquid containers2810 and2820, which in the some embodiments are about 10 milliliters each.Container2810 communicates withport350 viatube2850 andcontainer2820 communicates withport360 viatube2860. Aninlet port2812 and avent port2811 are located at or near the top ofcontainer2810.Container2820 also includes aninlet port2822 and avent2821 at or near its top.Port2812 ofcontainer2810 andport2822 ofcontainer2820 are both connected to avalve assembly2828 viavalves2840 and2841. Anagitator2801, which may be a nitrogen gas (N₂) or other gas, is connected tovalve assembly2828 by fitting2851.Valves2840 and2841 regulate the flow of N₂into their respective containers. In some embodiments, additional containers (not shown) may be provided, similar tocontainer2810, for introducing a buffer and/or other fluid into the cavity.
• In operation, a fluid is placed into[0167]container2810. The fluid, for example, may contain targets that are to be hybridized with probes on the chip.Container2810 is sealed by closingport2811 whilecontainer2820 is vented by openingport2821. Next, N₂is injected intocontainer2810, forcing the fluid throughtube2850,cavity310, and finally intocontainer2820. The bubbles formed by the N₂agitate the fluid as it circulates through the system. When the amount of fluid incontainer2810 nears empty, the system reverses the flow of the fluid by closingvalve2840 andport2821 andopening valve2841 andport2811. This cycle is repeated until the reaction between the probes and targets is completed.
• In some applications, foaming may occur when N[0168]₂interacts with the fluid. Foaming potentially inhibits the flow of the fluid through the system. To alleviate this problem, a detergent such as CTAB may be added to the fluid. In one embodiment, the amount of CTAB added is about 1 millimolar. Additionally, the CTAB affects the probes and targets positively by increasing the rate at which they bind, thus decreasing the reaction time required.
• The system described in FIG. 28 may be operated in an alternative manner. According to this technique, back pressure formed in the second container is used to reverse the flow of the solution. In operation, the fluid is placed in[0169]container2810 and bothports2811 and2821 are closed. As N₂is injected intocontainer2810, the fluid is forced throughtube2850,cavity310, and finally intocontainer2820. Because the vent port incontainer2820 is closed, the pressure therein begins to build as the volume of fluid and N₂increases. When the amount of fluid incontainer2810 nears empty, the flow of N₂intocontainer2810 is terminated by closingvalve2840. Next, the circulatory system is vented by openingport2811 ofcontainer2810. As a result, the pressure incontainer2820 forces the solution back through the system towardcontainer2810. In one embodiment, the system is injected with N₂for about 3 seconds and vented for about 3 seconds. This cycle is repeated until hybridization between the probes and targets is completed.
• FIG. 29 illustrates an alternative embodiment of the agitation system.[0170]System2900 includes avortexer2910 on which thechip package300 is mounted. Acontainer2930 for holding the fluid communicates withinlet350 viatube2950. Avalve2935 may be provided to control the flow of solution into the cavity. In some embodiments,circulator2901, which may be a N₂source or other gas source, is connected tocontainer2930. Alternatively, a pump or other fluid transfer device may be employed. The flow of N₂intocontainer2930 is regulated by avalve2936.Circulator2901 is also connected toinlet tube2950 via avalve2902.
• A[0171]waste container2920 communicates withport360 viaoutlet tube2955. In one embodiment, aliquid sensor2940 may be provided for sensing the presence of liquid inoutlet tube2955. Access to the waste container may be controlled by avalve2921. Optionally, additional containers (not shown), similar tocontainer2930, may be employed for introducing a buffer or other fluid into the cavity.
• The system is initialized by closing all valves and filling[0172]container2930 with, for example, a fluid containing targets. Next,valves2936,2935, and2955 are opened. This allows N₂to entercontainer2930 which forces the fluid to flow throughtube2950 and into the cavity. When the cavity is filled,valves2935,2936, and2955 are closed to seal the fluid in the cavity. Next, the vortexer is activated to vibrate the chip package, similar to a paint mixer. In some embodiments, the vortexer may vibrate the package at about 3000 cycles per minutes. The motion mixes the targets in the fluid, shortening the incubation period. In some embodiments, the vortexer rotates the chip package until hybridization is completed. Upon completion,valve2902 and2955 are opened to allow N₂into the cavity. The N₂empties the fluid intowaste container2920. Subsequently, the cavity may be filled with a buffer or other fluid.
• FIG. 30 illustrates an alternative embodiment in which the agitation system is partially integrated into the chip package. As shown,[0173]chip package300 includes acavity310 on which the chip is mounted.Cavity310 is provided withinlets360 and350. The package also includeschambers3010 and3020. Aport3021 is provided inchamber3010 and is connected toinlet360 by achannel3025.
• [0174]Chamber3010 is equipped withports3011 and3012.Port3012 communicates withinlet350 through achannel3015.Channel3015 is provided with awaste port3016 that communicates with afluid disposal system3500 via atube3501. Avalve3502 regulates the flow of fluids into the disposal system. In some embodiments, the disposal system includes awaste container3510 andfluid recovery container3520 which are connected totube3501. Avalve3530 is provided to direct the flow of fluids into either the waste container or recovery container.
• Port[0175]3011 is coupled to afluid delivery system3600 through atube3601. Fluids flowing intochamber3010 from the fluid delivery system are regulated by avalve3602. The fluid delivery system includesfluid containers3610 and3620 that are interconnected with atube3690.Container3610, which may hold a fluid containing targets, includesports3616 and3615.Port3616 is connected totube3690. Avalve3612 controls the flow of the fluid out ofcontainer3610. Acirculator3605, which may be a N₂source, is connected toport3615 ofcontainer3610. Alternatively, any type of gas, pump or other fluid transfer device may be employed. The flow of N₂intocontainer3610 is controlled by avalve3618. Avalve3619 may also be provided to ventcontainer3610.
• [0176]Container3620, which may hold a buffer, is provided withports3625 and3626.Circulator3605 is connected toport3625. Avalve3621 is provided to control the flow of N₂intocontainer3620.Port3626 is connected totube3690 via avalve3622.Valve3622 regulates the flow of the buffer out ofcontainer3620. Optionally, additional containers (not shown), similar tocontainer3620, may be configured for introducing other fluids into the cavity. Avalve3690 connects circulator3605 totube3690 for controlling the flow of N₂directly into the package. Avalve3652 is provided for venting the fluid delivery system.
• In the initial operating state, all valves are shut. To start the hybridization process, a fluid containing targets is introduced into chamber[0177]301 by openingvalves3602,3612 and3618. This injects N₂intocontainer3610 which forces the fluid to flow through3601 and intochamber3010. Whenchamber3010 is filled,valves3612 and3618 are closed. Next,valve3642 is opened, allowing N₂to flow directly intochamber3010. The N₂agitates and circulates the fluid intocavity310 and out tochamber3020. As the volume of fluid and N₂inchamber3020 increase, likewise does the pressure therein. Whenchamber3020 approaches its capacity,valve3642 is closed to stop the fluid flow. Thereafter, the system is vented by openingvalve3652. Venting the system allows the back pressure inchamber3020 to reverse the flow of fluids back intochamber3010. Whenchamber3010 is filled,valve3652 is closed andvalve3642 is opened to reverse the fluid flow. This cycle is repeated until hybridization is completed.
• When hybridization is completed, the system may be drained. This procedure depends on which chamber the fluid is located in. If the fluid is located in[0178]chamber3020, thenvalve3502 is opened, whilevalve3530 is positioned to direct the fluid into the appropriate container (recovery or waste). The pressure inchamber3020 forces the fluid throughport3016,tube3501, and into the disposal system. If the fluid is inchamber3010, thenvalve3502 and3642 are opened. As a result, N₂forces the fluid inchamber3010 throughport3501 and into the disposal system.
• Once the system is emptied, all valves are closed. A buffer or other fluid may be introduced into the cavity. For example, the cavity may be filled with a buffer by opening[0179]valves3601,3621, and3622. This injects N₂intocontainer3620 which forces the buffer therein to flow through the system until it fillscavity310. In the alternative, ultrasonic radiation, heat, magnetic beads, or other agitation techniques may be employed.
• The present inventions provide commercially feasible devices for packaging a probe chip. It is to be understood that the above description is intended to be illustrative and not restrictive. Many embodiments will be apparent to those skilled in the art upon reviewing the above description. Merely as an example, the package may be molded or machined from a single piece of material instead of two. Also, other asymmetrical designs may be employed to orient the package onto the detection systems.[0180]
• The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents.[0181]

Claims (92)

What is claimed is:

1. A method of making probe chips comprising the steps of:

forming a plurality of probe arrays on a substrate;

separating said substrate into a plurality of chips, each of said chips comprising at least one probe array thereon; and

mating at least one of said chips to a package, said package comprising a reaction chamber, said reaction chamber comprising inlets for flowing fluid therein, said at least one probe array in fluid communication with said reaction chamber.

2. The method as recited inclaim 1 wherein said package is made by the steps of:

injection molding first and second halves of said package; and

mating said first and second halves together.

3. The method as recited inclaim 2 wherein one of said halves comprises flow channels therein, said flow channels in communication with said inlets.

4. The method as recited inclaim 3 further comprising the step of applying a reenterable seal to flow channels in said package.

5. The method as recited inclaim 1 wherein said substrate comprises alignment marks for forming said probe arrays thereon in a desired position, and wherein said alignment marks are used to identify locations for said separating of said substrate into chips.

6. The method as recited inclaim 1 wherein said package comprises an alignment structure thereon, wherein said step of mating said chip to said package uses said alignment structures to position said package at a desired position.

7. The method as recited inclaim 1 wherein said package comprises an alignment structure thereon, and further comprising the step of identifying the location of at least one target on said probe array in a scanner, wherein said package is placed at a desired location in said scanner using said alignment structure.

8. The method as recited inclaim 1 wherein said step of forming a plurality of probe arrays comprises the steps of:

selectively exposing said substrate to light;

coupling selected monomers to said substrate where said substrate has been exposed to light.

9. The method as recited inclaim 1 wherein said step of separating comprises the steps of:

scribing said substrate in desired locations;

breaking said substrate along said scribe lines.

10. The method as recited inclaim 1 wherein said step of forming a plurality of probe arrays on said substrate is a step of forming a plurality of oligonucleotide probe arrays on said substrate.

11. The method as recited inclaim 10 further comprising the steps of flowing labeled oligonucleotide target molecules through said reaction chamber and identifying where said target molecules have bound to said substrate.

12. The method as recited inclaim 11 wherein said package comprises a temperature probe and further comprising the step of monitoring and adjusting a temperature in said reaction chamber.

13. The method as recited inclaim 1 wherein said package is formed by the steps of:

forming first and second package portions; and

acoustically welding said first and second package portions together.

14. The method as recited inclaim 1 wherein said step of mating said chips to packages comprises the step of binding said chips to said package with an adhesive.

15. The method as recited inclaim 14 wherein said packages comprise a recessed region thereon, whereby said chips do not extend above a surface of said packages.

16. The method as recited inclaim 1 further comprising the step of flowing target molecules through said reaction chamber.

17. An apparatus for packaging a substrate, said apparatus comprising:

a substrate having a first surface and a second surface, said first surface comprising a probe array;

a body having a mounting surface with a fluid cavity, said second surface attached to said cavity; and

a cover attached to said mounting surface for sealing said cavity.

18. The apparatus ofclaim 17 wherein said cavity comprises an inlet port and an outlet port, said inlet and outlet ports permitting fluids to circulate into and through said cavity.

19. The apparatus ofclaim 18 wherein said inlet and outlet ports comprise a reenterable seal.

20. The apparatus ofclaim 17 wherein said probe array comprises an array of oligonucleotide probes.

21. An apparatus for packaging a substrate, said apparatus comprising:

a substrate having a first surface and a second surface, said first surface comprising a probe array and said second surface being an outer periphery of said first surface;

a body having a mounting surface, an upper surface, and a cavity bounded by said mounting surface and said upper surface, said second surface being attached to said cavity and said first surface being within said cavity; and

a cover attached to said mounting surface for defining an upper boundary to said cavity;

wherein said cavity comprises a diffuser and a concentrator, said diffuser and said concentrator permitting laminar fluid flow through said cavity.

22. The apparatus ofclaim 21 wherein said probe array comprises an array of oligonucleotide probes.

23. The apparatus ofclaim 21 wherein said cover comprises a depression for receiving a temperature control element to maintain a reaction temperature in said cavity.

24. The apparatus ofclaim 21 wherein said cover comprises a first half mated to a second half.

25. The apparatus ofclaim 24 wherein said first half comprises a first channel and a second channel, said first channel being in fluid communication with said diffuser and said second channel being in fluid communication with said concentrator.

26. The apparatus ofclaim 25 wherein said second half comprises a third channel and a fourth channel, said third channel being in fluid communication with said first channel, and said fourth channel being in fluid communication with said second channel. .

27. The apparatus ofclaim 26 wherein said first channel and said second channel comprise re-enterable seals for sealing fluid in said cavity.

28. An apparatus for mixing a fluid, the apparatus comprising:

a first substrate comprising a first inner surface functionalized with a microarray of reactive moieties;

a substantially parallel second substrate also comprising a second inner surface, wherein said first and second inner surfaces bound a closed chamber there between, said chamber adapted to retain a quantity of fluid so that the fluid is in contact with both surfaces;

at least one bubble disposed within said chamber; and

means for moving the chamber so that the bubble moves relative to the fluid to effect mixing of the fluid.

29. An apparatus for mixing a fluid, the apparatus comprising:

a first substrate comprising a first inner surface functionalized with a microarray of reactive moieties;

a substantially parallel second substrate also comprising a second inner surface, wherein said first and second inner surfaces bound a closed chamber there between, said chamber adapted to retain a quantity of fluid so that the fluid is in contact with both surfaces;

at least one bubble disposed within said chamber, wherein said bubble is a magnetic particle; and

means for moving the bubble relative to the fluid to effect mixing of the fluid.

30. The apparatus ofclaim 28, wherein the closed chamber has a thickness of less than about 2 millimeters.

31. The apparatus ofclaim 29, wherein the closed chamber has a thickness of less than about 2 millimeters.

32. The apparatus ofclaim 28, wherein both inner surfaces are functionalized with reactive moieties.

33. The apparatus ofclaim 29, wherein both inner surfaces are functionalized with reactive moieties.

34. The apparatus ofclaim 28, wherein the bubble comprises a gas.

35. The apparatus ofclaim 28, wherein the bubble comprises nitrogen.

36. The apparatus ofclaim 29, wherein said magnetic particle is a magnetic bead.

37. The apparatus ofclaim 28, wherein the bubble is produced by introducing a volume of the fluid that is less than the total volume of the closed chamber.

38. The apparatus ofclaim 28, further including a flexible seal between the inner surface of the first substrate and the inner surface of the second substrate.

39. The apparatus ofclaim 38, wherein said flexible seal includes a gasket.

40. The apparatus ofclaim 29, further including a flexible seal between the inner surface of the first substrate and the inner surface of the second substrate.

41. The apparatus ofclaim 40, wherein said flexible seal includes a gasket.

42. The apparatus ofclaim 28, further comprising means for introducing fluid into the closed chamber.

43. The apparatus ofclaim 29, further comprising means for introducing fluid into the closed chamber.

44. The apparatus ofclaim 28, wherein the first substrate and the second substrate are individually comprised of a material selected from the group consisting of glass, silicon, fused silica, plastic, and a combination thereof.

45. The apparatus ofclaim 29, wherein the first substrate and the second substrate are individually comprised of a material selected from the group consisting of glass, silicon, fused silica, plastic, and a combination thereof.

46. The apparatus ofclaim 28, wherein the first substrate is comprised of glass.

47. The apparatus ofclaim 29, wherein the first substrate is comprised of glass.

48. The apparatus ofclaim 28, wherein the means for moving the bubble is selected from the group consisting of rotating the apparatus about an axis, rolling the apparatus, and reciprocally shaking the apparatus.

49. A method for mixing a fluid, comprising:

providing an apparatus according toclaim 28;

introducing a fluid into the closed chamber;

introducing a bubble within the fluid; and

moving the bubble in the fluid to effect mixing of the fluid.

50. A method for mixing a fluid, comprising:

providing an apparatus according toclaim 29;

introducing a fluid into the closed chamber;

introducing a bubble within the fluid; and

moving the bubble in the fluid to effect mixing of the fluid.

51. A method for mixing a fluid, comprising:

providing an apparatus according toclaim 30;

introducing a fluid into the closed chamber,

introducing a bubble within the fluid; and

moving the bubble in the fluid to effect mixing of the fluid.

52. A method for mixing a fluid, comprising:

providing an apparatus according toclaim 31;

introducing a fluid into the closed chamber;

introducing a bubble within the fluid; and

moving the bubble in the fluid to effect mixing of the fluid.

53. A method for mixing a fluid, comprising:

providing an apparatus according toclaim 36;

introducing a fluid into the closed chamber;

introducing a bubble within the fluid; and

moving the bubble in the fluid to effect mixing of the fluid.

54. A method for mixing a fluid, comprising:

providing an apparatus according toclaim 48;

introducing a fluid into the closed chamber;

introducing a bubble within the fluid; and

moving the bubble in the fluid to effect mixing of the fluid.

55. An apparatus for mixing a fluid, comprising:

a first substrate and a second substrate having inner surfaces that define a closed chamber therebetween, said chamber adapted to retain a quantity of fluid so that the fluid is in contact with both inner surfaces;

means for creating bubbles in the fluid within the apparatus, whereby each bubble displaces the fluid resulting in mixing; and

means for moving a bubble in the fluid.

56. The apparatus ofclaim 55, wherein the first substrate comprises a material selected from the group consisting of glass, silicon, fused silica, and plastic.

57. An apparatus for mixing a fluid, comprising:

a first substrate and a substantially parallel second substrate having inner surfaces that define a closed chamber therebetween, said chamber adapted to retain a quantity of fluid so that the fluid is in contact with both inner surfaces;

means for providing bubbles in the fluid within the apparatus, whereby each said bubble displaces the fluid resulting in mixing; and

means for moving a bubble in the fluid.

58. An apparatus for mixing a fluid, comprising:

a first substrate and a second substrate having inner surfaces that define a closed chamber therebetween, said chamber adapted to retain a quantity of fluid so that the fluid is in contact with both inner surfaces; and

means for creating bubbles in the fluid at selected locations within the apparatus, whereby each bubble displaces the fluid resulting in mixing; and wherein at least one of said inner surfaces is functionalized with reactive moieties.

59. The apparatus ofclaim 58 wherein the reactive moieties comprise monomeric species covalently bound to said inner surface, each of the monomeric species having at least one reactive site.

60. The apparatus ofclaim 59 wherein the monomeric species are nucleotides.

61. The apparatus ofclaim 60 wherein the monomeric species are amino acids.

62. The apparatus ofclaim 61 wherein the reactive moieties comprise reactive sites of monomeric species present at the terminus of a surface-bound polymer.

63. The apparatus ofclaim 62 wherein the surface-bound polymer comprises a polynucleotide.

64. The apparatus ofclaim 62 wherein the surface-bound polymer comprises a polyribonucleotide.

65. The apparatus ofclaim 64, wherein the surface-bound polymer comprises a polypeptide.

66. A method comprising:

providing a first substrate and a second substrate having inner surfaces that define a closed chamber therebetween, said chamber adapted to retain a quantity of fluid so that the fluid is in contact with both inner surfaces, and wherein at least one of said inner surfaces is functionalized with polynucleotides, polypeptides, or polysaccharides;

introducing a fluid containing a plurality of components into the closed chamber so as to provide a quantity of fluid therein in contact with both inner surfaces;

providing a bubble in the fluid; and

moving a bubble within the fluid to result in mixing.

67. A method according toclaim 66, wherein the polynucleotide is a polyribonucleotide.

68. A method according toclaim 66, wherein the chamber is adapted to retain a film of fluid in contact with both inner surfaces.

69. A method according toclaim 66 wherein the inner surfaces of the first and second substrates are substantially parallel.

70. A method according toclaim 66, wherein the chamber is less than two millimeters in thickness.

71. A method according toclaim 66 further including using heat for said mixing.

72. A method according toclaim 66 further including using ultrasonic radiation for said mixing.

73. A method ofclaim 66, wherein the at least one of said inner surfaces is functionalized with polynucleotides.

74. A method of66, wherein the at least one of said inner surfaces is functionalized with polypeptides.

75. A method comprising:

providing a first substrate and a second substrate having inner surfaces that define a closed chamber therebetween, said chamber adapted to retain a quantity of fluid so that the fluid is in contact with both inner surfaces, and wherein at least one of said inner surfaces is functionalized with an array of RNA or DNA probes;

introducing a fluid sample containing DNA or RNA into the closed chamber so as to provide a quantity of fluid therein in contact with both inner surfaces;

providing a bubble in the fluid;

moving a bubble within the fluid to result in mixing;

after hybridization is complete, removing the sample from the apparatus; and

analyzing the functionalized inner surface for DNA or RNA that has hybridized.

76. A method according toclaim 75 additionally comprising heating the DNA or RNA containing sample fluid while in the closed chamber.

77. A method according toclaim 76 additionally comprising washing the functionalized inner surface prior to the analyzing.

78. A method according toclaim 75, wherein the bubble is moved in a circular pattern.

79. A method according toclaim 78, wherein the bubble is moved in the circular pattern that includes exiting the closed chamber.

80. A method comprising:

providing a first substrate and a second substrate having inner surfaces that define a closed chamber therebetween, said chamber adapted to retain a quantity of fluid so that the fluid is in contact with both inner surfaces, and wherein at least one of said inner surfaces is functionalized with an immobilized biological polymer;

introducing a fluid containing a plurality of components into the closed chamber so as to provide a quantity of fluid therein in contact with both inner surfaces;

providing a bubble in the fluid; and

moving a bubble within the fluid to result in mixing.

81. A method according toclaim 80, wherein said biological polymer includes a polynucleotide.

82. A method according toclaim 81, wherein said polynucleotide is a polyribonucleotide.

83. A method according toclaim 80, wherein said biological polymer includes polypeptides.

84. A method according toclaim 80, wherein said biological polymer includes polysaccharides.

85. A method for mixing a film of fluid, comprising:

providing a first substrate and a substantially parallel second substrate having inner surfaces that define a closed chamber therebetween, said chamber adapted to retain a quantity of fluid so that the fluid is in contact with both inner surfaces;

introducing a fluid containing a plurality of components into the closed chamber so as to provide a film of fluid therein; and

nucleating a bubble within the film of fluid, whereby, as the bubble is nucleated and dispelled, the fluid is displaced resulting in mixing.

86. The method ofclaim 85, wherein the dispelling comprises moving the bubble.

87. An apparatus for mixing a fluid, comprising:

a first substrate and a second substrate having inner surfaces that define a closed chamber therebetween, said chamber adapted to retain a quantity of fluid so that the fluid is in contact with both inner surfaces;

means for nucleating bubbles in the fluid comprising discrete sources for creating individual bubbles at selected locations within the apparatus, whereby, as each bubble is nucleated and dispelled, the fluid is displaced resulting in mixing; and

means for moving a bubble in the fluid.

88. The apparatus ofclaim 87, wherein the means for moving a bubble includes a pump.

89. An apparatus for mixing a fluid, comprising:

a first substrate and a substantially parallel second substrate having inner surfaces that define a closed chamber therebetween, said chamber adapted to retain a quantity of fluid so that the fluid is in contact with both inner surfaces;

means for nucleating bubbles in the fluid comprising discrete sources for creating individual bubbles at selected locations within the apparatus, whereby, as each bubble is nucleated and dispelled, the fluid is displaced resulting in mixing; and

means for moving a bubble in the fluid.

90. The apparatus ofclaim 89, wherein the means for moving a bubble includes a pump.

91. An apparatus for mixing a fluid, comprising:

a first substrate and a substantially parallel second substrate having inner surfaces that define a closed chamber therebetween, said chamber adapted to retain a quantity of fluid so that the fluid is in contact with both inner surfaces; and

means for nucleating bubbles in the fluid comprising discrete, heat sources for creating individual bubbles at selected locations within the apparatus, whereby, as each bubble is nucleated and dispelled, the fluid is displaced resulting in mixing; and wherein said means for nucleating bubbles also comprises means for moving a bubble in the fluid.

92. The apparatus ofclaim 91, wherein the means for moving a bubble includes a pump.

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