Patent Application
20080066634
The present invention relates to printing devices, and more particularly, to a microcontact printing device and a microcontact printhead containing one or more of the microcontact printing devices.
The microarray format for preparing samples of biological materials is the primary method used for monitoring gene expression and several other important biological parameters. In current microarray formats, arrays of approximately 500 nm -200 μm spots of DNA, RNA, proteins or other biological samples are deposited onto a glass substrate using microcontact printing devices including sharpened stainless steel needles or pins. In a typical experiment, between 4 and 64 of the steel pins are dipped into wells of a source plate each well of which contains a different DNA sample, and then touched to the substrate to deposit a spot of DNA. The spots are subsequently subjected to a hybridization reaction with probe/target DNA samples to determine the relative amounts of various DNA molecules in the sample.
The stainless steel pins are typically fabricated from 1/16″ stainless steel rod stock with the sharp tip and capillary channel-fluid reservoir spark cut one at a time with EDM (electronic discharge machine). This laborious serial process results in a current sales price of the pins from $175-625/pin. Recent additions of laser cut and electropolished pins are similarly priced.
In addition to cost issues, the current technology used to fabricate microarrays has other weaknesses. Variability exists in the DNA deposits due to poor pin-to-pin uniformity of printing tip geometry and the sample volume deposited, which leads to difficulties in analysis and decreased confidence in results. The range of DNA deposit sizes that can be printed is currently limited by current printing tip designs, however, it would be advantageous to fit more deposits into smaller spacing on the glass surface. The current technology wastes precious DNA samples, because only a percentage of the sample imbibed into the pin is actually transferred to the glass surface. The chemical resistance and mechanical strength of the pins is an issue as is the fact that the printing tips tend to wear and deform which leads to variability in deposit characteristics. The printing pressure of the pins is merely controlled by gravity as there is no mechanism for controlling printing pressure. The only way the pins can be filled with a sample is by dipping In addition, the steel pins have a limited uptake volume which is often less than 1 μL.
Accordingly, a microcontact printing device is needed that addresses the problems associated with current microcontact printing devices.
According to a first aspect of the disclosure, a microcontact printing device comprising a tube member for storing or transferring a printing fluid or liquid, and a printing element attached to an end of the fluid dispensing member.
According to another aspect of the disclosure, a microcontact printhead device comprising a holder and at least one microcontact printing device disposed within the holder, the microcontact printing device including a tube member for storing or transferring a printing fluid or liquid and a printing element attached to an end of the fluid dispensing member.
According to a further aspect of the disclosure, a method of fabricating a microcontact printing device comprising steps of providing a wafer or substrate, micromachining a printing element from the wafer or substrate, providing a tube member for storing or transferring a printing fluid or liquid, and attaching the printing element to an end of the tube member.
A first aspect of the disclosure is a microcontact printing device. The microcontact printing device is especially useful for printing and manufacturing high quality microarrays of proteins, DNA, RNA, polypeptides, oligonucleotides and microarrays of other biological materials having spot volumes in the range of 10−10 picoliters to 100 nanoliters. The microcontact printing device may also be used for printing and manufacturing high quality microarrays of other matters including, without limitation, solid semiconductor quantum dots or liquid dots containing various functional molecules, such as sensors, organic small molecules, organic polymers, solutions of organic polymers, dyes, inks, adhesives, molten metals, solders, glasses, and ceramic oxides.
Referring now to the drawings and initially to
The tube member 20 may be made of any suitable material or combination of materials including, but not limited to, glasses, polymers, metals, metal alloys, ceramics, and silicon. In some embodiments, the tube member 20 may comprise a length of glass or polymer tubing. The glass or polymer tubing may be rigid or flexible, straight or curved. The tube member 20 may include, but is not limited to, round cylindrical outer and inner surfaces 20o and 20i, respectively. It is preferred that the inner surface 20i of the tube member 20 be a round cylindrical surface or some other cylindrical surface shape that avoids sharp corners to provide a smooth flow of a fluid/liquid therethrough, as sharp corners tend to entrain the fluid/liquid and interrupt the fluid flow. Other possible inner cylindrical surface 20i shapes include, without limitation, oval, hexagonal, octagonal, and irregular shapes. The outer cylindrical surface 20o may be other shapes including, without limitation, square, rectangular, oval, hexagonal, octagonal, and irregular shapes. In a preferred embodiment, the tube member 20 comprises a Pyrex® glass tube with round cylindrical outer and inner surfaces 20o and 20i, an outer diameter of about 2 mm, and an inner diameter of about 1 mm. The outer and inner dimensions of the tube member 20 are, of course, not limited to those provided above.
In some embodiments, the fluid conduit 26 of the tube member 20 (best seen in
In one embodiment, as shown in
In other embodiments, the force and compliance necessary for successful printing is not provided by tethers, but by a continuous membrane whose thickness, flexibility and elasticity are chosen to provide the required degree of compliance (if any) in a direction perpendicular to the plane of the substrate.
In a preferred embodiment, the perimeter frame 32, the printing tip 40 and the one or more tethers 50 of the printing element 30 are formed as a single unitary member. It is also contemplated that one or more of the perimeter frame 32, the printing tip 40 and the one or more tethers 50 of the printing element 30 may be formed separately and then attached to the other components of the printing element 30 using any suitable bonding technique, in alternate embodiments. The printing element 30 with its perimeter frame 32, printing tip 40 and one or more tethers 50, whether unitarily or separately formed, may be made of any material or combination of materials that are suitable for microfabrication including, without limitation, silicon (Si), silicon oxides (SiO2), germanium (Ge), germanium-silicon (Ge—Si) alloys, silicon carbide (SiC), silicon nitride (Si3N4), polymers, ceramics, ferric alloys, and non-ferric alloys. Any suitable microfabrication method or combination of methods may be used for making the components of the printing element 30, depending upon the material or materials selected therefor, the desired dimensional precision of the printing element 30 and/or the desired manufacturing yield. Suitable microfabrication methods include but are not limited to chemical and physical microfabrication, photolithography, photoresist methods, micro-electromechanical methods, e-beam lithography, and x-ray lithography. Precision machining techniques including, without limitation, EDM, drilling and laser cutting techniques, may be used to supplement the microfabrication methods. The printing element 30 may be micromachined as a single unitary member, as mentioned earlier, from a substrate or wafer made of, but not limited to, a semiconductor, ceramic, glass, a metallic, and polymer materials, using conventional photolithographic, wet etching, and Deep Reactive Ion Etching (DRIE) techniques, as will be explained further on. The DRIE process allows hundreds or thousands of individual printing elements 30 to be formed in bulk from a single wafer or substrate. One or more of the individual components of the printing element 30 may also be formed from one or more of the earlier mentioned substrates or wafers or combination of substrates or wafers.
Referring to
The fluid dispensing channel 42 extends longitudinally through the printing tip 40 and communicates with the fluid conduit 26 of the tube member 20 at the bottom end 22 thereof. A fluid/liquid outlet 44 is defined by the fluid dispensing channel 42 at a free end of the printing tip 40. The free end of the print tip 40 also defines a rim surface 46 that contacts a substrate to be printed on during printing. In some embodiments, the rim surface 46 may be a substantially flat surface. In other embodiments, the rim surface 46 may be a concave or convex surface. The surface finish of the rim surface 46 may be smooth, textured or undulating. The rim surface 46 in some embodiments may be oriented generally perpendicular to the axis of the fluid dispensing channel 42. In still other embodiments, the rim surface 46 may be formed by multiple surfaces disposed at various angles to the fluid dispensing channel 42.
Referring to
When the rim surface 46 of the printing tip 40 contacts the substrate during printing, a small amount of the printing fluid/liquid is dispensed onto the substrate in a manner similar to that of a quill or a fountain pen, i.e., the substrate removes the fluid/liquid from the fluid/liquid filled fluid conduit 26 of the printing device 10.
Referring again to
If the one or more flexible tethers 50 are too thin, the printing tip 40 may sag, have insufficient mechanical stability, possess increased lateral motion when the printing tip 40 contacts the substrate and/or be subject to low frequency resonant modes. If the flexible tethers 50 are too thick, the printing tip 40 will not be able to deflect over the large required vertical/z displacement, which in one embodiment may be about 200μ of vertical/z displacement, without breakage. In some embodiments, to avoid breakage of the printing element 30 when the printing tip 40 is forced into the substrate along the z direction, the printing element 30 should be sufficiently flexible to allow the printing tip 40 and the one or more flexible tethers 50 to deflect completely up into the fluid conduit 26 of the tube member 20. In one embodiment, each of the four tethers 50 may have a thickness or height of about 30μ and a median width W of about 70μ. The vertical/z displacement requirement, e.g., 200μ of deflection, is very demanding given the lateral area of the printing element 30. The spiral-shape of the flexible one or more tethers 50 increases their effective length and thus, allows the stress of the deflection to be spread out over a longer distance. Although most substrates are locally flat to within 2-10μ, variations in z, over very large platters (up to about 1 meter2) which deliver the slides under the microcontact printhead device in a typical microarray printing station, can be easily this large.
In other embodiments where breakage may not be of a concern or a problem, the tethers 50 may be made substantially rigid.
The one or more tethers 50 are also constructed in a manner that utilizes capillary forces to direct the printing fluid/liquid into the fluid dispensing channel 42 of the printing tip 40 during printing. This may be accomplished by progressively decreasing the gap G (
In an alternative embodiment, the one or more tethers may also be of a constant width and provided with lateral, interdigital texturing, as described and shown in U.S. patent application Ser. No. 10/795,188, entitled MICROCONTACT PRINTHEAD DEVICE, now U.S. Patent Application Publication No. 20040233250, which is incorporated herein by reference. The lateral, interdigital texturing is provided on both sides of each tether on the portion of the tether closest to the printing tip. Moving further away from the printing tip, the lateral, interdigital texturing is provided only on the side of the tether facing towards the printing tip. The portions of the tethers most remote from the printing tip are not provided with the lateral, interdigital texturing.
The increased surface area provided by the lateral, interdigital texturing also enables the tethers to utilize capillary forces to direct the printing fluid/liquid into the fluid dispending channel of the printing tip during printing. The increased surface area provided by the lateral, interdigital texturing also prevents the printing fluid/liquid from flowing through the gaps between the one or more tethers or tether portions.
In a preferred embodiment, the printing element 30 including the one or more flexible tethers 50 are fabricated from silicon (a single crystal silicon substrate). In such an embodiment, the one or more flexible tethers 50 of the printing element 30 will virtually never fatigue because of the elastic properties of silicon and the lack of crystal grain boundaries in the single crystal silicon substrate. Unlike metal springs, the one or more silicon flexible tethers 50 will virtually always return the printing tip 30 to the same position and will deflect with the same amount of force during each printing cycle.
Another aspect of the disclosure is a microcontact printhead device.
Another aspect of the present invention is a method of fabricating the microcontact printing device 10. The printing element 30 of the microcontact printing device is preferably made from silicon using silicon micromachining methods. Silicon micromachining refers to the selective removal of defined regions of silicon or masking material, on the length scales of millimeters to nanometers, from a silicon substrate by an etching process. Etching is the primary means by which the third dimension of a micromachined structure is obtained from a planar photolithographic process. In the case of the printing element 30, the perimeter frame, the printing tip 40, the one or more flexible tethers 50 are all three dimensional structures. There are generally two main types of anisotropic etching processes: anisotropic wet etching using hot aqueous KOH and dry/plasma etching techniques such as DRIE. For both etching techniques, the pattern to be etched is defined by a photolithographic process. The silicon substrate from which the printing element 30 will be fabricated is preferably a single crystal silicon wafer, usually with a (100) orientation. The anisotropic wet etching technique involves, after patterned removal of the etch resistant silicon dioxide outer layer, etching at approximately 80° C. in aqueous KOH. Ethylenediamine may also be used as a wet etchant. This chemical etch attacks the silicon <100> planes many times faster than the <111> planes and can be used to etch square pits with approximately 57° <111> sidewalls into (100) silicon wafers. One advantage of the wet etching technique is that many wafers may be inexpensively etched in parallel. A disadvantage of the wet etching technique is that it only cuts along certain crystallographic planes and not at arbitrary angles. The most selective dry etching technique is DRIE, which is noted for its ability to etch very high aspect ratio trenches. This plasma technique rapidly pulses the etchant and passivator gasses alternatively over the substrate. DRIE is capable of cutting a thin approximately 10-20μ wide trench through a 500μ thick wafer with sidewalls vertical to within a few degrees over the depth of the cut. The pattern to be etched is simply defined in photoresist, which etches much more slowly than the silicon, and the etch removes the silicon not protected by the etch-resistant photoresist. An advantage of DRIE is that any arbitrary shape can be cut to very high precision but a potential disadvantage is that only one wafer at a time can be processed.
As shown in
As shown in
As shown in
The wafer 202 may then be thermally oxidized to form a coating of SiO2 over the printing element(s) 30 (in embodiments where the wafer is made of silicon) and separated from the wafer 200, as shown in
The microcontact printing device disclosed herein addresses the deficiencies of conventional steel-based pins. The DRIE process, which may be used for fabricating the printing elements of the printing device, produces cuts approximately 100× more precise and smooth than the techniques used to fabricate conventional steel-based machine shop pins. In addition, the DRIE process allows hundreds of printing elements to be fabricated in parallel, thus, pin-to-pin variation is essentially eliminated as compared to the steel pins. The higher micromachining precision also results in far more uniform printing tip rim surface, which yields more consistently shaped spots and is capable of producing a printing tip rim surface having a printing surface area of between about 5×107 and 10−6 square micrometers. Further, both the printing device density in the microcontact printhead device disclosed herein and the size of the printing tips can be easily miniaturized. Approximately 20 μm diameter printing tips on about 50 μm to about 125 μm centers or less, may be achieved using the fabrication method disclosed herein. Accordingly a microcontact printhead device having a printing tip or printing element density between about 2 and 1012 printing tips/elements per square centimeter may be achieved. Because of their construction, it is not possible to pack the conventional steel pins closer than the 4.5 mm spacing of the 384 format. Printing tips/elements on 50 μm centers are approximately 8×103 denser than the densities of steel pins in conventional holders. Since the printing elements in the preferred embodiment are made of silicon, a thin silicon dioxide (SiO2) film typically coats the surfaces of the printing elements. The wetting properties, chemical compatibilities and derivatization chemistry of SiO2 are well known as compared to the Cr2O3 surface of conventional stainless steel pins. Moreover, silicon is harder and much more elastic than stainless steel and will therefore wear much more slowly. The microcontact printhead device is also much less costly to manufacture than conventional steel pin microcontact printing devices.
A further aspect of the disclosure is a method of printing a microarray using the microcontact printhead device disclosed herein. In one embodiment of the printing method, a different solution of a sample of a DNA oligonucleotide, for example DNA in 3×SSC buffer, is dispensed into the fluid conduits of the printing devices mounted in the microcontact printhead device using an active fluid transfer device, such as a manual or automated pipetting system, e.g., liquid handling robot or pipette. A substrate is then prepared for printing by coating a flat glass microscope slide with a reagent to immobilize the DNA. The reagent may be polylysine or other protonated surface amino group. The microcontact printing devices of the microcontact printhead device are quickly touched to the substrate surface with a force sufficient to cause each printing tip to deposit a small quantity, including without limitation, 10−10 picoliters to 100 nanoliters, of the DNA printing fluid onto the substrate. The substrate with the DNA microarray of deposited spots may then be used for experiments, such as gene expression monitoring by subjecting the microarray to hybridization reactions.
The printing method, in another embodiment, comprises preparing a droplet array on a surface, using a solution of a sample of a DNA oligonucleotide, for example DNA in 3×SSC buffer, and controlling the atmosphere above the samples so that the samples do not evaporate. The droplet array may be prepared using, for example, an automated liquid dispenser, which places the droplets onto a patterned hydrophobic surface. The hydrophobic surface precludes any lateral movement of the droplet. The microcontact printing devices or the microcontact printhead device are loaded by dipping the printing tips of the printing devices into their corresponding droplets of the droplet array (instead of loading the printing fluid directly into the fluid conduits of the printing devices as in the previous embodiment). A substrate is then prepared for printing by coating a flat glass microscope slide with a reagent to immobilize the DNA. The reagent may be polylysine or other protonated surface amino group. The microcontact devices of the microcontact printhead device are quickly touched to the substrate surface with a force sufficient to cause each printing tip to deposit a small quantity of the DNA printing fluid onto the substrate. The small quantity of the DNA printing fluid deposited on the substrate may range between 10−10 picoliters to 10 nanoliters, 10−10. The substrate with the DNA microarray of deposited spots may then be used for experiments, such as gene expression monitoring by subjecting the microarray to hybridization reactions.
The micromachined microcontact printing devices disclosed herein address many of the shortcomings and needs of conventional microcontact printing devices. It is clear that users of microcontact printing technologies, and the DNA microarray fabrication process itself, can benefit from the precision, rapid prototyping and economy of scale of the microcontact printing devices disclosed herein. In addition, the microcontact printing devices may be readily adapted to existing printing hardware.
While the foregoing invention has been described with reference to the above, various modifications and changes can be made without departing from the spirit of the invention. Accordingly, all such modifications and changes are considered to be within the scope of the appended claims.
This Application claims the benefit of U.S. Provisional Patent Application No. 60/846,614 filed Sep. 22, 2006, the entire disclosure of which is incorporated by reference. This Application is related to U.S. patent application Ser. No. 10/220,913, entitled MICROFABRICATED SPOTTING APPARATUS FOR PRODUCING LOW COST MICROARRAYS, now U.S. patent application publication no. 20030166263; and U.S. patent application Ser. No. 10/795,188, entitled MICROCONTACT PRINTHEAD DEVICE, now U.S. Patent Application Publication No. 20040233250, the entire disclosures of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60846614 | Sep 2006 | US |
