WO2007087449A2

WO2007087449A2 - Fluid transfer and printing devices

Info

Publication number: WO2007087449A2
Application number: PCT/US2007/002317
Authority: WO
Inventors: Robert C. Haushalter; Srinivas Vetcha
Original assignee: Parallel Synthesis Technologies, Inc.
Priority date: 2006-01-25
Filing date: 2007-01-25
Publication date: 2007-08-02
Also published as: WO2007087449A3

Abstract

A printing and fluid transfer pin (100) including a reservoir section (102) and a printing tip section (104). The printing tip section may have a dispensing end disposed at a first end thereof. The pin may further have a longitudinal channel (118) disposed through the reservoir section and the printing tip section for transferring fluid from the reservoir section to the dispensing end of the printing tip section. A portion of the longitudinal channel disposed in the printing tip section may extend only partially through a thickness of the printing tip section. Also, methods for making the pin described above and printheads containing such pins.

Description

FLUID TRANSFER AND PRINTING DEVICES

RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 60/762,293, filed on January 25, 2006, the entire disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention relates to fluid transfer and printing. More particularly, the invention relates to devices and methods for fluid transfer and printing.

BACKGROUND OF THE INVENTION

[0003] Microarray technology is emerging as one of the principal and fundamental investigational tools for a very wide variety of biological problems. Although the preparation of DNA microarrays for use in many types of analysis is one of the main applications today, it is clear that the basic concept of easily obtaining huge amounts of data from a rapid and relatively simple-to-use platform is set to penetrate most areas of biology and may find comparably broad use in chemistry and material science. Such diverse areas of biology including, without limitation, genetics, population biology, immunology, rational drug design, genetic engineering and therapies, protein engineering, developmental biology and structural biology, would benefit from a rapid infusion of an inexpensive version of microarray technology. As with many other areas of technology, the true power of microarray technology will only become fully utilized when it is efficiently coupled to other related or complementary technology. For example, the coupling of an inexpensive, and easy to use microarray technology to amplification techniques may allow an almost "real time" look into the biochemical machinery and mechanisms of a single cell as a function of time after various biochemical challenges: [0004] In order to derive maximum benefit from a young technology area such as that of microarrays, the technology needs to be simple, inexpensive to purchase and use and be of reasonable physical size. For microarray technology, this translates into a system that should give better performance than the best current system, in a more compact format at a much lower price.

[0005] Many embodiments of microarray-based experiments involve the following basic, and common steps: after defining the question or problem to be addressed by the microarray based experiment, a sample is bound to a substrate, such as a glass slide treated with a reagent capable of covalently bonding the DNA to the glass substrate. The sample to be tested is then applied to the substrate.

[0006] There aTβ three common methods used for applying a sample to a substrate, each with its own compliment of advantages and disadvantages. Some of the more important parameters for various dispensing devices are summarized in Table I below.

TABLE I

a)

[0007] It is clear from the data in Table 1 that microspotting pins are a competitive technology in terms of speed, quality and cost. [0008] There are several prior art pin designs that modify and control the fluid flow both on the pin shaft and within the surface features fabricated on or in the pin shaft. Prior art pins may serve as microcontact printing tools by printing small spots onto a substrate when the pin is wetted with a printing fluid by fluid transfer devices by dipping the pin into fluid A to fill the pin shaft and reservoir with fluid A, and then immersing the pin shaft of the pin into fluid B thereby transferring fluid A into fluid B by providing sufficient time after immersion in fluid B, for fluid A to diffuse into fluid B.

10009J Pins for printing microarrays may be made from a variety of materials including metals, glasses, silicon, ceramics and polymers. Commonly known pin designs include solid pins, open capillary pins, hollow bore capillary pins and grooved ring pins.

[00101 A solid pin typically includes a solid pin shaft, usually with a round or rectangular cross section, and thus do not have reservoirs, channels, slots or grooves formed in the pin shaft. The main disadvantage of a solid pin is that it has no internal reservoir or other surface features and therefore, is capable of removing only a small amount of fluid from the printing fluid source vessel per dip. Therefore, the pin must be frequently returned to the source for refills, which slows down the printing process.

[0011] An open capillary pin, also known as a split pin or split quill pin, typically includes a reservoir formed by a slot-like capillary channel that passes all the way through the shaft and is open on two sides. The pin shaft may be round or rectangular in cross section. The function of the reservoir is to increase the volume of the fluid that can be held by the pin, which in turn, leads to fewer returns to the source material storage location thereby decreasing the time used for printing.

[0012] A hollow bore capillary pin typically includes a reservoir formed by a longitudinal closed channel formed in the pin shaft. The pin shaft may be round or rectangular in cross section.

[0013] A grooved ring pin typically includes one or more annular grooves formed in the outer surface of the pin shaft. Such pins are typically used in passive fluid transfer application. ' [0014] Although the above pins operate satisfactorily, there remains a need for pins that can print and transfer fluids with greater speed, quality and at a lower cost.

SUMMARY

[0015] A printing and fluid transfer pin is disclosed, comprising a reservoir section and a printing tip section. The printing tip section may have a dispensing end disposed at a first end thereof. The pin may further comprise a longitudinal channel disposed through the reservoir section and the printing tip section for transferring fluid from the reservoir section to the dispensing end of the printing tip section. A portion of the longitudinal channel disposed in the printing tip section may extend only partially through a thickness of the printing tip section.

[0016] An apparatus for printing and fluid transfer is also disclosed. The apparatus may comprise a pin having a reservoir section and a printing tip section. The printing tip section may have a dispensing end disposed at a first end thereof. The pin may further comprise a longitudinal channel disposed through the reservoir section and the printing tip section for transferring fluid from the reservoir section to the dispensing end of the printing tip section. A portion of the longitudinal channel disposed in the printing tip section may extend only partially through a thickness of the printing tip section.

[0017] A method of making a printing and fluid transfer pin is also disclosed. The printing and fluid transfer pin may have a reservoir section and a printing tip section, and the printing tip section may have a dispensing end disposed at a first end thereof. The pin may further comprise a longitudinal channel disposed through the reservoir section and the printing tip section for transferring fluid from the reservoir section to the dispensing end of the printing tip section. Further, a portion of the longitudinal channel disposed in the printing tip section may extend only partially through a thickness of the printing tip section. The method may comprise providing a substrate having first and second surfaces; performing a first etching step on the first surface; and performing a second etching step on the second surface;.wherein the first and second etching steps define the pin. [0018] A method is also disclosed comprising steps of: forming a positive mold of a printing and fluid transfer pin using a bulk micromachining process; forming a negative mold of the pin from the positive mold using an electroforming process; and forming the pin from a polymeric material in the negative mold, the pin having a reservoir section and a printing tip section, the printing tip section having a dispensing end disposed at a first end thereof, the pin further comprising a longitudinal channel disposed through the reservoir section and the printing tip section for transferring fluid from the reservoir section to the dispensing end of the printing tip section, wherein a portion of the longitudinal channel disposed in the printing tip section extends only partially through a thickness of the printing tip section.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIGS. IA- 1C collectively show an embodiment of a pin for fluid transfer and printing applications.

[0020] FIGS. 2A-2B are cross-sectiόnal views of three different embodiments of a groove-like tapered channel.

[0021] FIGS. 3 A and 3B collectively show another embodiment of the pin.

[0022] FIGS. 4A, 4B, 5A, 5B, 5C, 6, and 7 show further embodiments of the pin.

[0023] FIGS. 8A-8E illustrate the factors affecting passive fluid flow. . on certain shapes of hydrophilic and hydrophobic surfaces. . ,

[0024] FIGS. 9A-9L illustrate an embodiment of a DRIE method that may be used for fabricating the pins.

[0025] FIGS. 1 OA- 1 OF illustrate an alternate embodiment of a DRIE method that may be used for fabricating the pins. [0026] FIGS. 1 IA-I ID illustrate an embodiment of a micromachined silicon mold method that may be used for fabricating the pins.

[0027] FIG. 12 shows an embodiment of a pin formed by bonding two pins (each pin being similar to the pin shown in FIG. 6) together with the channels facing each other.

[0028] FIG. 13 are images showing that increasing the number of reservoirs increases the number of spots that can produced for each filling of the pin. Scanned microarray images of Cy 3 labeled 9-mers in 3x SSC printed from a single uptake volume using silicon pins with 75μm x 75μm pin tip size and having a) one reservoir channel, b) two reservoir channels and c) four reservoir channels. The spots are printed on a 180μm spot pitch and the width of the channels near the pin tips are lOμm.

[0029] FIG. 14 shows scanned microarray images of Cy3 labeled 9- mers in 3x SSC printed from a single uptake volume using silicon pins having a tip size of 75x75μm with various channel. The spots are printed on a 180μm spot pitch and the width and depth of the channels near the pin tips are a) 15μm x 75μm, b) lOμm x 35μm, and c) 2μm x 75μm. As can be seen from the above images the spot uniformity and number of spots increases with the decrease in channel size.

[0030] FIG. 15 shows scanned microarray images of Cy3 labeled 9- mers in 3x SSC printed from a single uptake volume using silicon pins with 75x75 μm pin tip size and having a) two partially etched reservoirs and b) two completely etched reservoirs. The spots are printed on an 180μm spot pitch and the width of the channels near the pin tips are lOμm.

[0031] FIG. 16 is an image showing that it is possible to obtain up to —2000 spots on a single fill of the pin. Block size: 49 x 42 spots, Spot pitch: 180μm, tip size: 75 x 75 μm², number of reservoirs: 4, channel width near tips: 2μm. [0032] FIG. 17 is a plot of spot size and coefficient of variance for groups of first 100, 200, 300 and 400 spots printed with three different types of pins all of which nominally have 75μm x 75μm print tips.

[0033] FIG. 18 shows a table comparing two pins of the present invention (PETC) with a split pin capillary design for microcontact printing applications. The data from this Table is plotted in FIG. 17.

[0034] FIG. 19A is a top view showing an embodiment of a printhead which contains an array of the pins of the present invention.

[0035] FIG. 19B is an elevational view showing of the printhead shown in FIG. 19 A.

[0036] FIG. 19C shows a microarray printed by the printhead collectively shown in FIGS. 19A and 19B

[0037] FIGS. 2OA and 2OB are cross sectional views showing other embodiments of the pin of the present invention formed by bonding two pins together with the channels facing opposite one another.

DETAILED DESCRIPTION OF THE INVENTION

[0038] Described below are pins for fluid transfer and printing applications. The pins may be used for transferring or printing any fluid that can be imbibed into the pin including without limitation proteins, DNA, RNA, polypeptides, oligonucleotides and other biological materials, solid semiconductor quantum dots or liquid dots containing various functional molecules, such as sensors, liquid metals and solder, and other materials.

[0039] FIGS. 1A-1C collectively show an embodiment of the pin for fluid transfer and printing applications, denoted by reference character 100. The pin 100 may include an elongated reservoir section 102, the first end of which is terminated with a tapered printing tip section 104. The reservoir and printing tip sections 102,104 define opposing first and second generally planar face surfaces 106,108 of the pin and opposing first and second generally planar lateral surfaces 110,112 of the pin 100. The pin 100 may have a generally rectangular or square profile when viewed transversely to a center line C_L of the pin 100. The printing tip section 104 defines a first end surface 114 of the pin 100 and the reservoir section 102 defines a second end surface 116 of the pin 100. The first end surface 114 of the pin 100 defined by the printing tip section 104 forms a dispensing surface or tip. The dispensing surface 114 is may be oriented generally perpendicular to the center line C_L of the pin 100, although inclined orientations are also contemplated. Further, the dispensing surface 114 may be a smooth planar surface as shown, a textured planar surface, an undulating surface, a concave surface, a convex surface, a multifaceted surface, and any combination thereof. In addition the dispensing surface 114 may be formed as an edge or as multiple edges. The second end 116 of the reservoir section 102 may be adapted for mounting in a pin mounting head (not shown).

[0040] The pin 100 further includes a longitudinal groove-like channel formed in the first surface 106 of the pin 100 that tapers toward of the printing tip section 104 (the channel or portions thereof also being referred to as a partially etched through channel or PETC when the pin is micromachined from a substrate using etching methods). The portion of the tapered channel 118 generally above the printing tip section 104 operates as a sample holding reservoir 120 and is referred to herein as a "partially open" sample holding reservoir 120. The tapered channel 114 communicates with the dispensing surface 114, thereby enabling a liquefied sample to be drawn into the tapered - channel 118 and stored in the partially open sample holding reservoir portion 120 thereof and then be dispensed at the dispensing surface 114 of the pin 100.

[0041] In one embodiment, the pin 100 may have a length Lp of about 28 mm, a width Wp of about lmm, and a thickness Tp about 400μ. The groove-like tapered channel 118 has a constant or variable depth Dc which is less than the thickness T_P of the pin 100 (FIGS. 2A-2C) along the length of the channel 118. Variable depth groove-like tapered channel 118 may include steps or undulations.

[0042] One of ordinary skill in the art will of course appreciate that other embodiments of the pin 100 including the embodiments described further on, may have dimensions other than those mentioned above. Moreover, in some embodiments, it is possible to provide a groove-like channel that does not taper.

[0043] As shown in FIG. 2 A, the groove-like tapered channel 118 may have an arcuate or curved profile 119 when viewed transversely to the center line CL of the pin (transverse profile). This profile 1 19 maximizes the fluid transfer efficiency of the channel 118 as there are no corners where the sample may tend to adhere to during transfer to the dispensing surface 114.

[0044] FIG. 2B shows an embodiment of the groove-like tapered channel having a generally V-shape transverse profile 1 19*. This channel profile is less efficient than the curved channel profile 119 shown in the embodiment of FIG. 2 A, but is more efficient than a channel having a rectangular- or square-shape transverse profile, because the V-shape transverse profile 119' has only one corner where the sample may adhere to rather than two corners, as in the rectangular- or square-shape transverse profile. The corner 118a of the channel 118 may be rounded as shown in FIG. 2B, to reduce sample adhesion in the corner 118a.

[0045] FIG. 2C shows an embodiment of the groove-like tapered channel 118 having a generally rectangular shape transverse profile 119" with rounded corners 118b. This channel profile 119" is more efficient than a rectangular or square transverse profile, because the corners 118bare rounded rather than sharp, thus, reducing sample adhesion in the corners 118b.

[0046] FIGS. 3 A and 3B collectively show another embodiment of the pin for fluid transfer and printing applications, denoted by reference character 200. Pin 200 shown in FIGS. 3A and 3B is similar to pin 100 shown in FIGS. IA, IB, and 2A-2C, except that the portion of the tapered channel 118 defined in the reservoir section 106 extends entirely through the reservoir section 102 from the first face surface 106 to the second face surface 108 to form an open sample holding reservoir 120'. The open sample holding reservoir 120' provides greater sample holding capacity than the partially closed sample holding reservoir 120 of pin 100 shown in FIGS. IA and IB. This, in turn, increases the amount of fluid that can be drawn into the pin 200, thereby increasing the number of spots that can be printed with each filling of the pin 200. The portion 121 of the tapered channel 118 defined in the printing tip section 104 is formed as a groove similar to the portion of the tapered channel 118 defined in the printing tip section 104 of pin 100 in FIGS. IA, IB, and 2A-2C.

[0047] FIG.4A shows a further embodiment of the pin for fluid transfer and printing applications, denoted by reference character 300. Pin 300 shown in FIG. 4 A is similar to pin 100 shown in FIGS. IA, IB, and 2A- 2C, except that a portion of the tapered channel 118 defined in the reservoir section 102 has an enlarged section 122 which increases the sample holding capacity of the reservoir and operates as a primary sample holding reservoir. In some embodiments, the enlarged section 122 of the reservoir may have a generally elliptical shape. The sample holding capacity can be increased even further by forming the portion of the tapered channel 118' extending between the enlarged channel section 122 and the printing tip section 104, in a winding or serpentine shape, as shown in FIG. 4B.

[0048] FIG. 5 A shows another embodiment of the pin for fluid transfer and printing applications, denoted by reference character 400. Pin 400 shown in FIG. 5A is similar to pin 100 shown in FIGS. IA, IB, and 2A-2C, except that includes two or more of the earlier described tapered channels 118. As shown in FIG. 5C the portions 121 of the tapered channels 118 extending through the printing tip section 104 of the pin 400 turn or are bent toward the center line CL of the pin so that they all terminate at and communicate with the dispensing surface 1 14. The multiple tapered channels 1 18 provided in the first face surface 106 of the pin 400 substantially increase the amount of fluid that can be drawn into the pin 400, thus, increasing the number of spots that can be printed with each filling of the pin 400. In addition, the multiple channels 118 reduce or eliminate missing spots during printing that may occur in a single channel pin if the channel 118 becomes clogged.

[0049] As shown in FIG. 5 B, the printing tip portions of the multiple channels 118 can be interconnected with each other by one or more groove- like lateral channels 124. The lateral channels 124 aid in maintaining the volume of a printed spot if one or more of the channels 118 becomes clogged, as fluid can be drawn from the other channels 118 that are not clogged through the lateral channel(s) 124.

[0050] FIG. 6 shows yet another embodiment of the pin for fluid transfer and printing applications, denoted by reference character 500. Pin 500 shown in FIG. 6 is similar to pin 100 shown in FIGS. IA₅ IB₅ and 2A-2C, except that the second face surface 108' is stepped such that the thickness of the reservoir section 102 of the pin 500 is greater than the thickness of the printing tip section 104 of the pin 500. This allows the partially closed reservoir 120 of the channel 118 to have a greater depth than the portion 121 of the channel 118 in the printing tip section 104 of the pin 600, thereby increasing the sample holding capacity of the partially closed sample holding reservoir 120.

[0051] FIG. 7 shows still another embodiment of the pin for fluid transfer and printing applications, denoted by reference character 600. Pin 600 shown in FIG. 7 is similar to pin 200 shown in FIGS. 3A and 3B, except that the second face surface 108* is stepped such that the thickness of the reservoir section 102 of the pin 600 is greater than the thickness of the printing tip 104 section of the pin 600. This provides pin 600 with a larger open sample holding reservoir 121" than pin 200, thereby increasing the sample holding ^; capacity of pin 600.

[0052] The channels 118 including their open or partially closed reservoir portions of all of the embodiments of the present invention disclosed herein may include the taper in the width thereof. In the example of the pin made of silicon (the pins may also be made from other semiconductor materials, glasses, metals, ceramics, and any other material that can be micromachined), as will be described further on, because of capillary and wetting forces of the aqueous or other solutions on a very wettable oxide Siθ2 surface of the silicon pin, as the sample of the printing fluid is consumed, the remaining solution is drawn by the taper in the reservoir and channel portion in the printing tip section. In other words, the thinner the capillary, the farther a column of liquid will be drawn into it by capillary forces. Therefore this taper causes all of the print fluid entrained in the pins' shaft to be delivered to the dispensing surface for subsequent transfer to the substrate as the print fluid is consumed. This taper is illustrated, along with other features important in passively directing fluid flow on and in the pin shaft, in FIG. 8.

[0053] FIG. 8 illustrates the factors affecting passive fluid flow on certain shapes of hydrophilic and hydrophobic surfaces. Liquid (in this example water) is drawn up into a hydrophilic tube but is pushed out of a hydrophobic tube when dipped into the liquid in FIG. 8A. The narrower the tube or capillary into which the liquid is drawn, the stronger the capillary forces, and the further the liquid is pulled into the tube in FIG. 8B. In FIG. 8C, the effect of the direction of the taper in the channel exiting into the print tip determines if the liquid will be drawn toward the tip, which results in the consumption of all print fluid imbibed into the channel being released via the printing protocols and procedures, is shown. When the channel tapers toward the tip FIG. 8C right all of the print fluid can be printed but when the taper is in the opposite direction FIG. 8C left, the liquid retreats up the shaft in the direction of the taper as the print fluid is withdrawn from the print tip and the pin concomitantly ceases to print. Pins possessing ridges on the reservoir walls as shown in FIG. 8D can imbibe more liquid than their corresponding smooth walled counterparts as shown in FIG. 8E.

[0054] In one embodiment, the pins of the present invention may be made of silicon and fabricated from silicon wafers using conventional silicon micromachining methods such as photolithography, wet etching, and Deep Reactive Ion Etching (DRIE). Silicon micromachining generally involves coating a silicon (Si) wafer to be micromachined with a masking material and patterning the masking material using photolithography followed by selective removal of regions of the Si wafer not covered by the patterned masking material using an etching method. Etching is the primary means by which the third dimension of a micromachined structure is obtained from a planar photolithographic method. There are generally two main types of etching methods used for micromachining, namely wet etching and dry/plasma etching. For both etching methods, the pattern to be etched is defined by a photolithographic method. The silicon pins are fabricated from a single crystal Si wafer that, in one embodiment, has a (100) orientation.

[00551 In one embodiment, the wet etching method involves: a) oxidation of a Si wafer to provide an SiO₂ layer, b) forming a photoresist layer over the SiO₂ layer using a spin coating technique and patterning the photoresist using photolithography, c) etching the exposed portion of the SiO₂ layer using a fluoride based etch (also known as a Buffered Oxide Etch) to expose the silicon beneath the SiO₂ layer and, d) etching the exposed silicon at approximately 80⁰C in aqueous KOH. The KOH etch attacks the silicon <100> planes many times faster than the <111> planes and may be used to etch square pits with 54.7° <111> sidewalls into the (100) Si wafer. The SiO₂ layer serves as an etch stop (hard mask) for the KOH etch process. A primary advantage of the wet etching method is that many wafers can be inexpensively etched in parallel. Wet etching, however, only etches along certain crystallographic planes and not at arbitrary angles.

[0056] The most selective dry/plasma etching method is Deep Reactive Ion Etching (DRIE), which is noted for its ability to etch features with very high aspect ratios. This plasma based method rapidly pulses etchant and passivator gasses alternatively over the Si wafer. The: wafer to be etched is oxidized and coated with a photoresist using a spin coating process. The pattern to be etched is defined in photoresist and the SiO₂ layer is selectively etched by a fluoride based etch. Both the photoresist and SiO∑ layers serve as etch stops in the DRIE method, as both the layers etch slower than silicon. Hence, either a SiO₂ layer or a photoresist layer or both can be used as etch stops in the DRIE method. The etch removes the portions of the Si wafer not masked by the etch-resistant SiO₂ and/or photoresist. By employing DRIE method, it is possible to make cuts perpendicular to the surface of the Si wafer in an anisotropic fashion and form channels having a depth : width ratio (aspect ratio) of 10 or more with nearly vertical sidewalls. Essentially any arbitrary shape can be cut into the silicon in this manner limited only by the resolution of the photolithographic process, or, as in this case, by how narrow and deep a channel can be cut by the DRIE method.

[0057] In one embodiment, the pins of the present invention may be made of silicon using a multi-step etch method comprising either a combination of wet KOH etching and DRIE.

[0058] In another embodiment, the pins of the present invention may be made of silicon using a multi-step etch method that uses only DRIE. In one embodiment of the DRIE method, which includes three DRIE operations (the three DRIE step method), the entire outline of the pin, channel, and tip are cut by DRIE in a first side of a Si wafer by patterning with two photomasks and using two DRIE steps. The different etch depths for the channel and the pin outline are achieved by patterning the photoresist layer and the Siθ₂ layer with the two different photomasks, where the photoresist and SiO₂ layers serve separately as etch stops for two different DRIE etching steps and by controlling the relative DRIE etch depths for each of the patterned layers. A second side of the Si wafer is patterned using a third photomask which is aligned to the etch on the first side of the wafer by standard double sided mask alignment techniques, using infrared radiation to "see" through the wafer. The pattern on the second side of the wafer produced by the third photo mask contains the outline of the pin, the portion of the channel for creating the open sample holding reservoir (this feature would be omitted før the pins with the partially closed sample holding reservoir), as well as the area to be thinned on the tip to reduce its size. The wafer is then etched on the second side using one DRIE step. By controlling the relative etch depths on the first and second sides of the wafer, the desired tip thickness can be obtained using the same photomask

[0059] The three DRIE step method commences by oxidizing a Si wafer to form an SiO₂ mask layer on a first side of the wafer and spin coating a first layer of photoresist on the SiO₂ mask layer. In one embodiment the Si wafer may be a 200μm thick <100> Si wafer. The first layer of photoresist is patterned with photomask A as shown in FIG. 9A and developed. The portions of the Siθ₂ mask layer not covered by the first layer of photoresist are then removed using, for example, a buffer oxide etch.

[0060] In FIG. 9B, the patterned first layer of photoresist is removed to expose the etched Siθ₂ mask layer (hatched area). The first side of the wafer is spin coated with a second layer of photoresist and the second layer of photoresist is patterned with photomask 9B (solid area) as shown in FIG. 9C.

[0061] In FIG. 9D, the wafer is etched using a first DRIE operation. The second layer of photoresist is then removed to expose the patterned Siθ2 layer, as shown in FIG. 9E. In FIG. 9F, the first side of the Si wafer is etched using a second DRIE operation.

[0062] The first SiO₂ mask layer on the first side of the Si wafer is stripped. The Si wafer is then flipped upside down and reoxidized to form a second SiO₂ mask layer on a second side of the Si wafer. A third layer of photoresist is formed over the second SiO₂ mask layer and is patterned with photomask C to form a pin with the open sample holding reservoir, as shown in FIG. 9G or with photomask D to form a pin with the partially closed sample holding reservoir, as shown in FIG. 9J. The second side of the Si wafer is etched using a third DRIE operation using the pattern second photoresist layer and the second SiO₂ mask layer as a mask, as shown in FIG. 9H (open sample holding reservoir) and FIG. 9K (partially closed sample holding reservoir). The Si wafer is then stripped of the third layer of photoresist and the second SiO₂ mask layer to provide the pin shown in FIG. 91 (open sample holding reservoir) or FIG. 9L (partially closed sample holding reservoir).

[0063] In one embodiment (starting with a 200 μm thick Si wafer), the first DRIE operation extends 50μm into the first side of the Si wafer and the second DRIE operation extends an additional 50μm into the first side of the Si wafer to provide a total etch depth of lOOμm in the first side of the Si wafer. The third DRIE operation in this embodiment extends lOOμm into the second side of the Si wafer. Accordingly, a pin with a 100 x 100 μm² tip and 50μm deep channel in the printing tip section. The pin tip dimension (in the y direction, FIGS. 91 and 9L) and the channel depth in the printing tip section can be made in any desired size by varying the relative etch depths using the same photomasks.

[0064] In another embodiment of the DRIE method, which includes two DRIE operations (the two DRIE step method), the method is based on the fact that DRIE etch rate is slower in a narrow region where material is removed as compared to a wider region. Unlike the three DRIE step method where the channel and/or reservoir depth are controlled by etch stops using two different photomasks and two DRIE etch operations on the first side of the Si wafer, the two DRIE step method uses only one photomask and one DRIE operation to control the channel depth. In the two DRIE step method, the depth of the channel is controlled by the width of the channel on the photomask. By using sufficiently narrow channel widths (e.g., approximately <2μm) the channel depth is allowed to terminate naturally, as the etch rate inside the narrow channel tends to slow with respect to the etch rate at wider openings such as those near the pin outline. In other words, when the channel to be etched is sufficiently narrow, the diffusion rate of the etchant gas drops as the depth of the channel increases until eventually the diffusion rate required to etch the bottom of the channel has slowed to the point that the etching effectively stops and the channel terminates part way through the region to be etched. The maximum channel etch depth for a certain channel width is determined by the aspect ratio of the DRIE etching apparatus and is typically in the range of 1 : 10, i.e., a channel with 2 μm channel width can be etched up to ~ 20μm deep using a etcher having an aspect ratio of 1 : 10. .

[0065] The two DRIE step method commences by oxidizing a Si wafer to form an SiO₂ mask layer on a first side of the wafer and spin coating a first layer of photoresist on the SiO₂ mask layer. In one embodiment the Si wafer may be a 200μm thick <100> Si wafer. In FIG. 1OA, the first layer of photoresist is patterned with photomask M.

[0066] The first side of the Si wafer shown in FIG. 1OB is etched (e.g., lOOμrή deep) using a first DRIE operation. As can be seen in FIG. 1OC, the narrow channel adjacent the pin tip terminates without etching lOOμm deep while the pin outline and wider channels etch lOOμm deep.

[00671 The first SiO∑ mask layer and first layer of photoresist on the first side of the Si wafer is stripped. The Si wafer is then flipped upside down and reoxidized to form a second SiO₂ mask layer on a second side of the Si wafer. A second layer of photoresist is formed over the second SiO₂ mask layer and is patterned with photomask N₅ as shown in FIG. 12D, which will form a pin with a partially closed sample holding reservoir. The second side of the Si wafer is etched using a second DRIE operation (etched another lOOμm) using the patterned second photoresist layer and the second SiO₂ mask layer as a mask, as shown in FIG. 1OE. The Si wafer is then stripped of the second layer of photoresist and the second Siθ₂ mask layer to provide the pin shown in FIG 1OF. The channel depth can be controlled by the channel width in the Photomask M and the pin tip can be made any desired size by varying the relative etch depths on the two sides of the Si wafer.

[0068] In an alternative embodiment, the pins of the present invention with the open or partially closed sample holding reservoirs may be made of any suitable polymer. In one embodiment, such pins can be fabricated from a micromachined silicon mold. The steps include making a positive mold of the desired part in silicon (i.e. the same as the final part itself) onto which an electroformed mold is electrolytically deposited using the micromachined silicon (which is suitably sensitized) as the cathode. The electroformed mold, in one embodiment, may be made of a Co-Ni or Ni-Fe alloy. The silicon is removed from this negative electroform and the electroform is used to • - ■ compression mold, resin cast or emboss the. pin from a polymer. Silicon molds are very inexpensive to prepare and are capable of containing much finer features than molds prepared by traditional machining techniques.

[0069] FIGS. 1 IA-I ID illustrate the fabrication of a pin with the open or partially closed sample holding reservoir according to one embodiment of a micromachined silicon mold method.

[0070] In FIG. 1 IA, a blank Si wafer is provided. In FIG. 1 IB, the Si wafer is micromachined to prepare a Si master mold using bulk microtnachining methods. In FIG. 11C, a metal mold is formed in the Si master mold. The metal mold may be made of nickel-cobalt. In FIG. 1 ID, the polymer pins are then molded using the metal mold. Molding may be implemented using any suitable polymer forming method. In one embodiment, a resin casting technique where the polymer precursors and a polymerization catalyst are mixed and poured into the mold which may be heated to accelerate the reaction, as shown in FIG. 1 ID. Other polymer forming methods, such as compression molding, hot embossing, injection molding and the like may also be used.

[0071] The performance of pins made with the open and partially closed sample holding reservoirs described herein were evaluated by printing DNA microarrays and analyzing certain parameters of the microcontact printed spots which are indicative of the quality of the microarray. The samples were printed with micromachined silicon pins with various size channels and compared with prior art pins having channels that extend entirely through the pin shaft. The samples were printed with a printing solution consisting of cy3-labeled random 9-mers of DNA in 3X SSC onto glass slides that had been previously coated with l-amino-3-(trimethoxysiryl)-n-propane and scanned on an Axon® microarray scanner at a pixel resolution of lOμ x lOμ.

[0072] The data in FIGS. 13-16, shows that the pin designs described herein are capable of printing microarrays of a higher quality than pins with previous open capillary designs. For a given pin tip size, the pin designs described herein will deliver. larger number of spots and spots with greater spot-to-spot uniformity as compared to the open capillary pins.

[0073] As shown in FIG. 13, increasing the number of reservoirs increases the number of spots that can produced for each filling of the pin. Scanned microarray images of Cy3 labeled 9-mers in 3x SSC printed from a single uptake volume using silicon pins with 75μm x 75 μm pin tip size and having a) one reservoir channel, b) two reservoir channels and c) four reservoir channels. The spots are printed on a 180μm spot pitch and the width of the channels near the pin tips are lOμm. [0074] FIG. 14 shows scanned microarray images of Cy3 labeled 9- mers in 3x SSC printed from a single uptake volume using silicon pins having a tip size of 75x75 μm with various channel. The spots are printed on a 180μm spot pitch and the width and depth of the channels near the pin tips are a) 15μm x 75μm, b) lOμm x 35μm, and c) 2μm x 75μm. As can be seen from the above images the spot uniformity and number of spots increases with the decrease in channel size. See the plot shown in FIG. 17 which compares the spot size profile of the above three arrays.

[0075] FIG. 15 shows scanned microarray images of Gy 3 labeled 9- mers in 3x SSC printed from a single uptake volume using silicon pins with 75x75 μm pin tip size and having a) two partially etched reservoirs and b) two completely etched reservoirs. The spots are printed on an 180μm spot pitch and the width of the channels near the pin tips are lOμm.

[0076] As shown in FIG. 16, it is possible to obtain up to -2000 spots on a single fill of the pin. Block size: 49 x 42 spots, Spot pitch: 180μm, tip size: 75 x 75 μm², number of reservoirs: 4, channel width near tips: 2μm.

[0077] As described earlier, a variety of pin, channel and reservoir shapes can be fabricated. The shapes and dimensions of the pin features affect the size, number, shape and volume of the printed spots and thereby exert a pronounced affect on the spot size, spot-to-spot uniformity and the number of spots that can be printed per each dip of the pin into the source plate. One of the factors that influences the spot size and reproducibility is the width of the channel as it exits the pin shaft onto the print tip.

[0078] Several of the experiments described here show the substantial effect of the width of the channel as it exits onto the dispensing surface of the printing tip section. The narrow channel that exits onto the dispensing surface print tip acts in a fashion analogous to a flow restrictor in that it mediates the flow between the reservoir and the print tip. As shown in FIGS. 14, 16 and 17 and the Table shown in FIG. 18, the width of the channel has a dramatic effect on the number of spots that can be printed per source plate visit or per aspiration. . . • [00791 As shown in FIG. 13, the number of spots that can be printed per aspiration increases as the number of reservoirs in the pin shaft increases as expected. Increasing the number of reservoirs from one to two to four, increases the number of spots that can be printed from approximately 600 to 1000 to 2000 spots, respectively. The data in FIGS. 14, 17 and the Table shown in FIG. 18 show the dramatic effect on spot size induced by the size and shape of the channel as it exits onto the print tip. The smaller the channel dimension as it enters the print tip the smaller and more uniform the spots that are produced.

[0080] As expected, the open reservoirs give more spots per dip than the partially closed as shown in FIG. 15. In FIG. 16 the combination of four reservoirs and a 2μ channel can give up to ~2000 spots per source plate visit.

[0081] In another embodiment, two Si wafers can be thermally bonded to each other to form a pin where the entire channel is closed or the channel portion in the printing tip section is closed as shown in FIG. 12.

[0082] The presence of multiple reservoirs in the pin increases the total reservoir volume the pin can hold and helps the pin to deliver more number of spots for each uptake volume. The reservoir volume within a single channel can also be increased by changing the geometry/shape of the reservoir. For example, the earlier mentioned serpentine channel shown in FIG. 4B can hold more fluid compared to earlier mentioned linear channel shown in FIG. IA formed within a comparable area on the two pin shafts.

[0083] Although the invention has been described in terms of exemplary embodiments, it is not limited thereto. Rather, the appended claims should be construed broadly, to include other variants and^~embodiments of the invention, which may be made by those skilled in the art without departing from the scope and range of equivalents of the invention.

Claims

CLAIMS What is claimed is:

1. A printing and fluid transfer pin, comprising a reservoir section and a printing tip section; the printing tip section having a dispensing end disposed at a first end thereof; the pin further comprising a longitudinal channel disposed through the reservoir section and the printing tip section for transferring fluid from the reservoir section to the dispensing end of the printing tip section; wherein a portion of the longitudinal channel disposed in the printing tip section extends only partially through a thickness of the printing tip section.

2. The pin of claim 1, wherein a portion of the longitudinal channel disposed in the reservoir section extends only partially through a thickness of the reservoir section.

3. The pin of claim 1, wherein the portion of the longitudinal channel disposed in the reservoir section extends all the way through a thickness of the reservoir section.

4. The pin of claim 1, wherein the pin is made from a semiconductor material.

5. The pin of claim 4, wherein the semiconductor material is silicon.

6. The pin of claim 1, wherein the pin is made from a polymer material

7. The pin of claim 1, wherein the pin is made from a ceramic material.

8. The pin of claim 1 , wherein the channel is tapered such that a width of the longitudinal channel disposed in the reservoir section is greater than a width of the longitudinal channel disposed in the printing tip section.

9. The pin of claim 8, wherein the portion of the longitudinal channel disposed in the printing tip section is tapered.

10. The pin of claim 8, wherein the portion of the longitudinal channel disposed in the reservoir section is tapered

11. The pin of claim 8, wherein the longitudinal channel is continually tapered from the reservoir end to the printing tip end.

12. The pin of claim 1 , wherein at least a portion of the longitudinal channel has a rectilinear cross section.

13. The pin of claim 1 , wherein at least a portion of the longitudinal channel has a curvilinear cross section.

14. The pin of claim 1, wherein at least a portion of the longitudinal channel has a triangular cross section.

15. The pin of claim 1, further comprising a plurality of longitudinal channels disposed through the reservoir section and the printing tip section for transferring fluid from the reservoir section to the dispensing end of the printing tip section.

16. The pin of claim 15, wherein two of the plurality of longitudinal channels are disposed on opposite sides of the pin.

17. The pin of claim 15; wherein a plurality of longitudinal channels are disposed on opposite sides of the pin.

18. The pin of claim 15, wherein at least two of the plurality of longitudinal channels are connected by a lateral channel.

19. The pin of claim 18, wherein the lateral channel is disposed within the printing tip section.

20. The pin of claim 15, wherein the plurality of longitudinal channels converge at the dispensing end.

21. The pin of claim 1, wherein the longitudinal channel has a serpentine shape along at least a portion of a length of the channel.

22. The pin of claim 1 , wherein at least a portion of the longitudinal channel is defined by an axial bore disposed within the pin.

23. An apparatus for printing and fluid transfer, the apparatus comprising: a pin having a reservoir section and a printing tip section; the printing tip section having a dispensing end disposed at a first end thereof; the pin further comprising a longitudinal channel disposed through the reservoir section and the printing tip section for transferring fluid from the reservoir section to the dispensing end of the printing tip section; wherein a portion of the longitudinal channel disposed in the printing tip section extends only partially through a thickness of the printing tip section.

24. The apparatus of claim 23, further comprising a holder for holding the pin.

25. The apparatus of claim 24, wherein the holder further comprises a second member for collimating the pin.

26. The apparatus of claim 23, further comprising at least a second pin, the pins forming an array.

27. The apparatus of claim 26, wherein the array of pins comprises up to 100,000 pins.

28. The apparatus of claim 26, wherein the array of pins form a pin density between about 10^"4 and 10⁶ pins/mm².

29. The apparatus of claim 23, wherein the pin is configured to contain a predetermined volume of fluid, the predetermined volume being between about 0.1 mL and 10^"4 pL.

30. The apparatus of claim 23, wherein pin is capable of printing a spot having an of about 10 mm² and 10^"6 μm².

31. The apparatus of claim 23, wherein the pin further comprises a plurality of longitudinal channels disposed through the reservoir section and the printing tip section for transferring fluid from the reservoir section to the dispensing end of the printing tip section.

32. The apparatus of claim 31, wherein two of the plurality of longitudinal channels are disposed on opposite sides of the. pin.

33. The apparatus of claim 31 , wherein a plurality of longitudinal channels are disposed on opposite sides of the pin.

34. The apparatus of claim 31 , wherein at least two of the plurality of longitudinal channels are connected by a lateral channel.

35. The apparatus of claim 34, wherein the lateral channel is disposed within the printing tip section.

36. The apparatus in of claim 31 , wherein the plurality of longitudinal channels converge at the dispensing end.

37. The apparatus of claim 23, wherein the longitudinal channel has a serpentine shape along at least a portion of a length of the channel.

38. The apparatus of claim 23 , wherein at least a portion of the longitudinal channel is defined by an axial bore disposed within the pin.

39. A method of making a printing and fluid transfer pin having a reservoir section and a printing tip section, the printing tip section having a dispensing end disposed at a first end thereof, the pin further comprising a longitudinal channel disposed through the reservoir section and the printing tip section for transferring fluid from the reservoir section to the dispensing end of the printing tip section, wherein a portion of the longitudinal channel disposed in the printing tip section extends only partially through a thickness of the printing tip section, the method comprising: providing a substrate having first and second surfaces; performing a first etching step on the first surface; and performing a second etching step on the second surface; wherein the first and second etching steps define the pin.

40. The method of claim 39, wherein the first etching step comprises a deep reactive ion etching process.

41. The method of claim 39, further comprising performing a third etching step on the first surface, the third etching step comprising a deep reactive ion etch.

42. The method of claim 39, wherein the first etching step defines the longitudinal channel.

43. A method, comprising steps of: forming a positive mold of a printing and fluid transfer pin using a bulk micromachining process; forming a negative mold of the pin from the positive mold using an electroforming process; and forming the pin from a polymeric material in the negative mold, the pin having a reservoir section and a printing tip section, the printing tip section having a dispensing end disposed at a first end thereof, the pin further comprising a longitudinal channel disposed through the reservoir section and the printing tip section for transferring fluid from the reservoir section to the dispensing end of the printing tip section, wherein a portion of the longitudinal channel disposed in the printing tip section extends only partially through- a thickness of the printing tip section.

44. The method of claim 43, wherein the pin further comprises a second member for collimating the pin.

44. The method of claim 43, wherein the pin further comprises at least a second printing and fluid transfer pin, the pins forming an array.

45. The method of claim 44, wherein the array of pins comprises up to 100,000 pins.

46. The method of claim 44, wherein the array of pins form a pin density between about 10^ and 10⁶ pins/mm². ^• i ^{• •}

47- The method of claim 43, wherein the pin is configured to contain a predetermined volume of fluid, the predetermined volume comprising between about 0.1 mL and 10"* pL.

48. The method of claim 43, wherein pin is capable of printing a spot having an area of about 10 mm² and 10^"6 μm².

49. The method according to claim 43, wherein the polymeric material is selected from the group consisting of polycarbonates and polymethylmethacrylates, polyolefϊns, and polyetherketones.

50. The method according to claim 43, wherein the polymeric material comprises a thermoplastic polymer.