JP2003516748A - Apparatus and method for producing bioarray - Google Patents

Abstract

  (57) [Summary] The present invention relates to a system for bio-array fabrication and a method for dispensing a controlled amount of liquid on a surface. In particular, the invention includes bio-array fabrication systems and methods for the controlled delivery of small volumes of a reaction into or onto a known target.

Description

Detailed Description of the Invention

This application is based on the U.S. patent application filed on Dec. 17, 1999. S. S. N. 09/46
5,314, filed December 17, 1999 09 / 465,960, 19
It is a continuation application of 09 / 465,959 filed on December 17, 1999. All of these are incorporated herein by reference.

FIELD OF THE INVENTION The present invention relates to systems for bio-array fabrication and methods for delivering a controlled amount of liquid onto a surface. More particularly, the invention includes bio-array fabrication systems, and methods for performing controlled delivery of small volumes of reactive liquid into or on known targets.

BACKGROUND OF THE INVENTION Molecular biology is a constantly evolving field of research. Of great importance in the field of molecular biology is the detection and analysis of DNA, RNA, bacteria and proteins. It is currently predicted that there will be a large market for bio-chips (micro-array chips) in the diagnosis and treatment of diseases. It is envisioned that the day will come when doctors will be able to use bio-chips to immediately make genetic marker-based diagnoses in the office, without the need for a laboratory as an intermediate diagnostic facility. At present, the greatest importance and market for bio-chips lies in the field of genetics and pharmaceutical research, where thousands of genes can be analyzed simultaneously.

[0004] In general, a DNA chip has a glass, silicon, or a porous support medium containing microscopic pieces or biomolecules of a large number of biomolecules, such as immobilized DNA probe sequences aligned on a surface. It consists of a plastic wafer. These are used to identify specific disease genes and to facilitate drug discovery efforts. In the future, the increasing use of biomolecule-related sciences is expected to enable more personalized medicine, in particular, the design and use of customized treatments and therapies based on the genetic makeup of the patient.

At present, it is known that bio-chips, especially DNA chips, are based on a common manufacturing method. That is, the etching of silicon computer chips as currently used in the semiconductor industry. Bio for studying biomolecules
Of all the uses of chips, DNA research is the most mature. In a particular example, a process of photoactivated DNA probe synthesis is used to produce high concentration DNA chips (Affymetrix Corp.). Typically 20 mer DNA
A photolithographic mask level 80 is used to synthesize the probe. Alternatively, a DNA chip is produced using donor technology to place the purified, previously synthesized oligonucleotides in specific locations on the surface. The latter technique does not require photolithography and the purity of the probe array is much higher than that of the photoactivated probe synthesis process, thus requiring less overlapping probes. Another means for synthesizing DNA probes is by using micromirrors that allow more than 300,000 bits of DNA to be placed on the chip in just a few hours. Furthermore, the use of ink-jet printing is known. This uses high speed robotic equipment to print the DNA on the small squares of the glass to form an array.
These types of machines are capable of forming as many as 32,000 DMA molecules on a single chip.

Yet another method uses fiber optic bundles to create a chip capable of holding 50,000 different DNA fragments on a single chip, and DNA molecules on the surface of the chip. Included is the use of microelectronic chips that utilize electricity to couple.

For many types of applications, it is necessary to define very small amounts of fluid (liquid) on a surface. Currently, this is done by dropping a small amount of liquid through the hole by using some pressure to drop the liquid itself. Piezoelectric power may be applied to deliver a small volume (picoliter to nanoliter) of liquid. However, in some instances, piezoelectric power can cause unwanted changes in the properties of the fluid, leading to denaturation of biomolecules contained in the fluid. In some applications it is necessary to dispense a large number of different drops of liquid. In any situation where dozens, thousands, or tens of thousands of different liquids need to be dispensed, the time required to complete the cycle of filling, dispensing and cleaning the piezoelectric donating tip for each different liquid is extreme. Long. In some applications, less liquid may be needed.

Accordingly, it is an object of the present invention to provide an apparatus and method for delivering a controlled small volume of liquid onto a target.

SUMMARY OF THE INVENTION In accordance with the objects outlined above, the present invention provides at least one (and preferably a plurality of) holding wells and dipping interconnected by microfluidic channels in fluid communication. ) A device is provided that includes a fluid reservoir that includes a well. In some cases, the holding well group, the dip well group, and the channel group are integrally formed of a single semiconductor material. In some cases, the holding well group is formed around the silicon wafer, and the dip well group is formed on the center side. Dipwells may be formed in a two-dimensional array or in a silicon wafer at multiple planar levels. The holding well may be characterized as holding a constant, known amount of liquid with a viscosity, and the dip well may be characterized as holding a known amount of liquid with a constant viscosity. The channel transports a constant, viscous, known amount of liquid from at least one holding well to at least one dip well, and at least one microfluidic channel extends from at least one holding well to at least one dip well, and so on. The holding well and the dip well are placed in fluid communication with each other.

The elements of the fluid reservoir can be formed by selective etching or wet etching. In one aspect, the invention provides a method of making a fluid reservoir for use in a bio-array fabrication system. The method comprises providing a semiconductor material,
Forming an oxide on the top surface of the semiconductor material, forming an aluminum material on the top surface of the oxide in the region of at least one microfluidic channel,
Forming an oxide material on the top surface of the aluminum material, depositing a mask on the top surface of the oxide material, etching the oxide material to retain at least one having a defined shape Defining a well and at least one dip well (each of the at least one holding well and the at least one dip well is characterized by holding a constant viscous, known amount of liquid); Etching to define at least one microfluidic channel in fluid communication with at least one holding well and at least one dip well (at least one microfluidic channel from at least one holding well to at least one dip well). Characterized in that it transports a known amount of liquid), Including the stage. Optionally, the method includes chemical mechanical polishing (CM) in which excess aluminum material is removed to leave aluminum material in the etched channels prior to forming the oxide material on the top surface of the aluminum.
P) is utilized.

In a further aspect, the invention features a base; and a plurality of tips integrally formed with the base projecting from the base, holding a known amount of liquid having a constant viscosity under surface tension. And a stamp including a plurality of tips. Optionally, each tip can be formed into a two-dimensional array, and can be formed by selective etching, or isotropic etching techniques can be used to define hydrophilic and hydrophobic structures. Optionally, the tips are depth stopped to control the level of immersion of the tips using anisotropic etching techniques (or other techniques).
It is formed including. The tip can be formed with an oxide structure at the terminal point of the tip.
The stamp can have 30,000-50,000 tips at 1 cm locations.

In a further aspect, the invention provides a method of producing a stamp for controlled delivery of liquid. The method comprises providing a semiconductor material, depositing a mask on a top surface of the semiconductor material, etching the semiconductor material to define a plurality of tips having a defined shape (each of the plurality of tips). Characterized in that it holds a known amount of liquid of constant viscosity under surface tension)). Optionally, the method includes the step of depositing a thick oxide on the top surface of the semiconductor material prior to etching. The etching step may include etching the silicon material using an isotropic etching technique to form a structure that defines hydrophilic and hydrophobic portions.

In a further aspect, the invention provides a stamp comprising a plurality of tips formed on a surface; a plurality of dipwells, each of which holds a sensing liquid, complementary to the plurality of tips formed on the surface of the stamp. A fluid reservoir comprising: a fluid reservoir further comprising a plurality of microfluidic channels; a sensor plate comprising a surface on which a minute amount of sensing liquid is distributed by a plurality of tips once immersed in a dipwell containing a sensing liquid, And thus forming a bio-array on a sensor plate. The tip can be anisotropically etched to define a depth stop.

In a further aspect, the system includes a detector, such as an optical or electrical detector, on or near the sensor plate.

In a further aspect, the present invention provides a stamp formed of a silicon material and including a plurality of tips formed on a surface, the stamp further defining a depth stop; and a plurality of tips formed on the surface of the stamp. Complementary fluid reservoir containing a plurality of dip wells each holding a sensing liquid, the fluid reservoir further comprising a plurality of microfluidic channels in fluid communication with the plurality of holding wells; A sensor plate comprising a surface on which minute amounts of sensing liquid are distributed by the plurality of tips once dipped into the well, thus forming a bio-array on the sensor plate,
A bio-array fabrication system is provided.

In a further aspect, the invention provides a method of controlled delivery of liquid. The method provides a stamp formed of a silicon material and including a plurality of tips formed on a surface, the stamp further defining a depth stop; complementary to the plurality of tips formed on the surface of the stamp. Providing a fluid reservoir formed with a plurality of dip wells each holding a sensing liquid, the fluid reservoir further comprising a plurality of microfluidic channels in fluid communication with the plurality of holding wells; Providing a sensor plate containing; dipped multiple tips into a dip well containing the sensing solution;
Dispensing minute amounts of sensing liquid to the sensor plate by stamping on the surface of the sensor plate, thus forming a bio-array on the sensor plate. The array can be formed in a single stamping process step or in multiple steps.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a plan view of a fluid reservoir illustrating a plurality of dip wells, retention wells and microfluidic channels formed therein in accordance with the present invention.

2-5 schematically illustrate a side view and a plan view of steps utilized during the creation of a fluid reservoir, including the creation of microfluidic channels, according to the present invention.

6-10 schematically illustrate a side view of the stages utilized during the creation of the fluid reservoir of the second embodiment, including the creation of microfluidic channels, in accordance with the present invention.

11-13 illustrate, in simplified form, a side view of the stages utilized during the creation of a channel void, in accordance with the present invention. And

FIG. 14 illustrates a simplified cross-sectional view of a multi-layer fluid storage chip according to the present invention.

FIG. 15 is a simplified side view illustrating a stamp including multiple tips according to the present invention.

16-19 schematically illustrate side views of steps utilized during the production of a stamp including multiple tips according to the present invention.

FIG. 20 is a simplified side view illustrating a stamp and reservoir of a bio-array fabrication system according to the present invention.

FIG. 21 is a simplified side view illustrating the stamp and sensor plate area of the bio-array fabrication system according to the present invention.

FIG. 22 is an enlarged side view illustrating a plurality of tips forming a stamp of a bio-array fabrication system according to the present invention.

FIG. 23 is an enlarged side view illustrating a dip well formed in a fluid reservoir of a bio-array fabrication system according to the present invention.

FIG. 24 is a plan view of a fluid reservoir, illustrating therein the microfluidic channels of the bio-array fabrication system according to the present invention. And

FIG. 25 is a plan view of a sensor plate illustrating a plurality of bio-array sites of the present invention.

[0030]   FIG. 26 illustrates a "one element" system described herein.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an apparatus and method for delivering a small amount of reactive liquid to a surface to produce a biochip. In particular, devices and methods for forming an array of sensing liquids on a surface for use in diagnostic assays are disclosed. This controlled dispensing of small amounts of liquid has a large number of test sites, with more than 100 test sites reaching 1 million test sites, while maintaining cost and time efficiency in chip production. , A chip such as a DNA chip is produced.

In general, the present invention provides for rapid and inexpensive formation of biological array populations by
Several different embodiments are provided. In the preferred embodiment, the system comprises a "one-element" system. It comprises a dispenser substrate having a number of storage wells on one side, the storage wells being connected via offset microfluidic channels to a donor head on the opposite surface of the substrate. By controlling the size of the storage wells, the viscosity and surface tension of the reagents in the wells, and the length and diameter of the offset channels, fluids can be easily applied to various sensor plates based on capillary action and surface tension. it can.

Alternatively, the present invention provides a “two element” system. The first element includes a substrate that includes a fluid reservoir. The fluid reservoir has holding wells that serve as reservoirs and dip wells connected by channels. The second element comprises a stamping substrate having a donor tip group that matches the Dipwell group. Thus, an array of reagents can be created by dipping the stamp into a fluid reservoir and then contacting it with the sensor plate.

Accordingly, the present invention provides methods and compositions for making arrays of biological reagents.
As will be appreciated by those in the art, biological reagents can be a variety of different substances. In a preferred embodiment, the biological reagent is a nucleic acid or protein.

By “nucleic acid” or “oligonucleotide” or grammatical equivalents herein is meant at least two nucleotides covalently linked together. Nucleic acids of the invention generally include phosphodiester bonds, but in some cases include nucleic acid analogs that may have alternative backbones, such as phosphoramide (Be
aucage et al., Tetrahedoron 49 (10): 1925 (1993) and references therein, Letsinger, J. Org. Chem. 35: 3800 (1970), Sprinzl et al., Eur.
J. Biochem. 81: 579 (1977), Letsinger et al., Nucl. Acids Res. 14: 3487 (1
986), Sawai et al., Chem. Lett. 805 (1984), Letsinger et al., J. Am. Che.
m. Soc. 110: 4470 (1988), and Pauwels et al., Chemica Scripta 26: 141 9
1986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19: 1437 (1991).
, And US Pat. No. 5,644,048), phosphorodithioate (Briu et.
al., J. Am. Chem. Soc. 111: 2321 (1989), O-methyl phosphoramidite chain (Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxfo).
rd University Press), and peptide nucleic acid backbones and chains (Egholm, J.
Am. Chem. Soc. 114: 1895 (1992), Meier et al., Chem. Int. Ed. Engl., 3
1: 1008 (1992), Nielsen, Nature, 365: 566 (1993), Carlsson et al., Natur.
e 380: 207 (1996), all of which are incorporated by reference. Other similar nucleic acids include positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA,
92: 6097 (1995)), nonionic backbone (US Pat. No. 5,386,023, 5th).
, 637,684, 5,602,240, 5,216,141 and 4,469,863, Kiedrowshi et al., Angew. Chem. Intl. Ed. Englis.
h, 30: 423 (1991), Letsinger et al., J. Am. Chem. Soc., 110: 4470 (1988).
, Letsinger et al., Nucleoside & Nucleotide, 13: 1597 (1994), ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Researc
h ", YS Sanghui and P. Dan Cook, Chapters 2 and 3, Mesmaeker et al.,
Bioorganic & Medicinal Chem. Lett., 4: 395 (1994), Jeffs et al., J. Biomo
lecular NMR, 34:17 (1994), Tetrahedron Lett. 37: 743 (1996)) and non-ribose backbones such as US Pat. Nos. 5,235,033 and 5,034,506.
Issue, and ASC Symposium Series 580, "Carbohydrate Modifications
in Antisense Research ", YS Sanghui and P. Dan Cook, eds., chapters 6 and 7. Also included within the definition of nucleic acids are nucleic acids containing one or more carbocyclic saccharides. (Jenkins et al., Chem. Soc. Rev. (1995)
pp 169-176). Some nucleic acid analogs are reviewed by Rawls, C & E News June 2, 19
97 page 35. Nucleic acid analogs include "locked nucleic acid.
acid) ”is also included. All of these references are incorporated by reference. These modifications of the ribose-phosphate backbone may be made to facilitate the addition of electron transfer moieties or to increase the stability and half-life of the molecule in physiological environments.

As will be appreciated by those in the art, all of these nucleic acid analogs can be used in the present invention. In addition, mixtures of naturally occurring nucleic acids and analogs can be made. For example,
Similar structures may be used for the conductive oligomer or electron transfer moiety binding sites.
Alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs can be made.

As outlined herein, a nucleic acid can be single or double stranded as specified, or can include portions of both double stranded or single stranded sequence. The nucleic acid can be DNA, RNA, or hybrid, both genomic DNA and cDNA, where the nucleic acid is any combination of deoxyribo- and ribo-nucleotides,
And any combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, isoguanine and the like. As used herein, the term "nucleoside" includes nucleotides and nucleosides and nucleotide analogs, and modified nucleosides such as amino modified nucleosides. In addition, "nucleoside" includes non-naturally occurring analog structures. Thus, individual units of peptide nucleic acid, eg, each containing a base, are referred to herein as nucleosides.

As used herein, “protein” or grammatical equivalents means proteins, oligopeptides and peptides, derivatives and analogs, including protein and pseudopeptide structures, including non-naturally occurring amino acids and amino acid analogs. Including. The side chain is (
The configuration may be either R) or (S). In a preferred embodiment, the amino acids are in the (S) or L-configuration.

Furthermore, depending on the reaction and the array, the biological reagent can be a complete biomolecule or a subunit added during synthesis. For example, in the case of nucleic acids, the biological reagent can be full length nucleic acid attached to the sensor plate. Alternatively, the present invention can be used in the synthesis of arrays; stepwise arrays can be prepared, eg, by adding a first nucleotide in the traditional manner, washing if necessary, and then adding a second nucleotide, etc. Can be produced.

Alternatively, arrays not normally classified as “biological”, but of other materials such as parts of organic chemicals, can be made as well.

Thus, in a preferred embodiment, the present invention comprises a "one-element" liquid dispensing system that includes a first substrate. As will be appreciated by those in the art, the substrates outlined herein include silicon wafers, silicon dioxide, silicon nitride, silicon such as glass and quartz glass, gallium arsenide, indium phosphide, aluminum, ceramics, Polyimide, quartz, plastic, resin, polymethylmethacrylate, acrylic, polyethylene, polyethylene terephthalate, polycarbonate,
Polystyrene and other styrene copolymers, polypropylene, polymers containing polytetrafluoroethylene, superalloys, zirconium alloys, steel, gold, silver,
Copper, tungsten, molybdenum, tantalum, KOVAR, KEVLAR, KAP
It can be made from a variety of materials including, but not limited to, TON, MYLAR, brass, sapphire, and the like. The preferred embodiment utilizes silicon and ceramic materials, depending on the reagents utilized. As will be appreciated by those in the art, the material forming the substrate should be compatible with the reagents outlined herein.

In this embodiment, the substrate comprises two surfaces, as generally shown in FIG.
Opposite of each other as generally outlined herein. The first surface includes a plurality of storage wells. "Storage well" herein means a chamber for holding a fluid, as outlined herein. Wells can be of various sizes, depending on the density of the arrays and the number of arrays created in a single run.

The second surface of the substrate includes a plurality of dispensing heads (sometimes referred to herein as nibs or tips). As will be appreciated by those in the art, these can be arranged in a variety of ways and include a number of different geometrical arrangements as are known in the art. Typically,
A regular XY arrangement is made.

The storage well and the donor tip are connected via a microchannel, as shown in FIG. As will be appreciated by those skilled in the art, this can be done in a number of configurations; importantly, depending on the size of the reservoir, surface tension and viscosity of the fluid to be dispensed, and the combination of other factors, the fluid To allow controlled delivery of. Generally, as shown in FIG. 26, the wells and corresponding heads are “offset” so that there is a distance between them. This is especially true when the device is used to make a dense array; each tip is in close proximity on one surface to achieve high density, but the reservoir may be spread out on the other surface.

In a preferred embodiment, the one-element system is made using two polished silicon wafers; one hollowed with a microfluidic channel on one side and a donor head on the other, and the other one. The sheet has a reservoir. Alternatively, a single two-sided silicon wafer can be used; the channels can be created with a sacrificial Al layer coated with oxide, which Al layer is subsequently removed using a wet etch.

Once created, the reservoir is filled using standard techniques and one dispenser
Contact one or more sensor plates to form an array. As will be appreciated by those skilled in the art, the sensor plate can be constructed of a variety of materials, including glass, ceramics, plastics, etc., as outlined above for substrates. In some embodiments, the sensor plate is coated or pretreated with a chemically functional material or a material such as a hydrogel to bind the biological reagent (s) (which may be covalent). Will be possible.

In a further preferred embodiment, the present invention provides a two element system including a fluid reservoir and a tip stamp. The fluid reservoir comprises a substrate containing at least one, and preferably a plurality of holding wells and dip wells, each connected by a microfluidic channel, as described above.

A preferred embodiment is illustrated in Figures 1-14. FIG. 1 illustrates a fluid reservoir including a plurality of dip wells, retention wells and microfluidic channels formed as part thereof. And Figures 2-11 illustrate the steps of creating a fluid reservoir according to the present invention.

Referring now to FIG. 1, illustrated is a plan view of a fluid reservoir 10 illustrating an arrangement of a plurality of dip wells 12, a plurality of holding wells 14, and a plurality of microfluidic channels 16. is doing. The reservoir 14 is formed using standard semiconductor processing techniques. Moreover, the reservoir 10 is formed to include a plurality of wells 12 and 14, as is typical in the formation of semiconductor chips that include a plurality of fanned bond pads. In particular, as shown by the dashed line in FIG. 1, a holding well 14 that serves as a large fluid reservoir and is formed on the outer periphery of the fluid reservoir 10 and a dip well 12 formed on the center side of the fluid reservoir 10. A plurality of microfluidic channels 16 are formed to place and in fluid communication. Typically, the holding well 14 is 1
On the order of 00 microns, the dimensions of an actual Dipwell 12 array are on the order of a few microns to tens of microns.

Referring now to FIGS. 2-5, illustrated is a portion of the fluid reservoir 10, showing the stages of fabrication of the fluid reservoir 10 of the first embodiment. As illustrated in FIG. 2, the semiconductor material, in particular a silicon wafer, 20 has a top surface 21
Is provided clean prior to further deposition of material. It should be understood that this disclosure contemplates that any type of semiconductor material such as plastic, glass, etc. can be used to form the fluid reservoir 10. The surface 21 has an oxide layer 22 formed thereon, such as silicon dioxide. Next, Dipwell 1
2, aluminum material 2 in the region forming the holding well 14 and the channel 16
Deposit 4 layers. It should be appreciated that any other material that can be selectively etched away, such as metal or an insulating material, can be utilized as material 24. Next, a layer 26 of thick oxide or nitride material is grown on the surface of the silicon material 20 and aluminum material 24, thus for fluids retained or transported in wells 12 and 14 and channel 16. To create an inert surface.

Referring now to FIG. 3, illustrated in a side cross-sectional view from the opposite direction of FIG. 2 is a portion of fluid reservoir 10. As illustrated, a mask layer 28, such as photoresist, is deposited on top of oxide layer 26. As illustrated in FIG. 4, mask layer 28 provides an etch of oxide layer 26 to define retention well 14 and dip well 12. During fabrication, the oxide material 26 is etched to expose a portion of the aluminum material 24, as illustrated in FIG. The aluminum material 24 is then wet etched to define flow microfluidic channels 26 and place the holding well 14 and dip well 12 in fluid communication.

Referring now to FIG. 5, only a portion of the fluid reservoir 10 is illustrated in simplified form in a plan view, and specifically illustrated is one holding well 14, one 2 is a storage chip including a dip well 12 and a microfluidic channel 16 illustrated by a dashed line. It should be understood that in this embodiment, the fluid reservoir 10 includes a plurality of holding wells 14, dip wells 12 and microfluidic channels 16.

Referring now to FIGS. 6-10, illustrated is a portion of the fluid reservoir 10 ′, showing the stages of fabrication of the fluid reservoir 10 ′ of the second embodiment. . It should be noted that all elements similar to those illustrated in FIGS. 2-5 are indicated with similar numbers with a dash added to indicate a different implementation. As illustrated in Figure 6, a silicon wafer 20 'is provided with the top surface 21 cleaned prior to further deposition of material. Silicon wafer 20 'has channel 2 in it
5 is etched. Surface 21 ', including within channel 25, has an oxide layer 22', such as silicon dioxide, formed thereon.

Referring now to FIG. 7, a layer of aluminum material 24 ′ is then deposited on the surface 21 ′, including within the channel 25, and the aluminum material 24 ′ is dipped into the dip well 12 ′.
, Holding well 14 'and channel 16' are deposited over the entire area to be formed (currently under consideration). The aluminum material 24 'is then planarized using a chemical mechanical polishing (CMP) technique, leaving the material 24' deposited in the channel 25. As illustrated in FIG. 8, a next layer of thick oxide or nitride material 26 'is grown on the surface of the silicon material 20' and aluminum material 24 ', thus wells 12' and 14 '.
'As well as being retained in the channels 16' or creating a surface inert to the fluid being transported.

Referring now to FIG. 9, illustrated in opposite side cross-sections of FIGS. 6 and 7 is a fluid reservoir 10 ′. As illustrated, a mask layer 28 ', such as photoresist, is deposited on top of oxide layer 26'. As illustrated in FIG. 10, the mask layer 28 'results in etching of the oxide layer 26' to define the retention well 14 'and the dip well 12'. During fabrication, the oxide material 26 'is etched to expose the aluminum material 24', as illustrated in FIG. Next, the aluminum material 24 'is wet-etched to form the microfluidic channel 2
6'is defined and the holding well 14 'and the dip well 12' are placed in fluid communication.

Referring now to FIGS. 11-13, a simplified illustration of a cross-sectional view is
A long channel 16 is required and therefore the aluminum material contained in the channel 2
4 is each step of the method for producing the storage chip 10 when the wet etching of No. 4 is difficult. In this example, voids 30 are etched in the oxide 26 along the length of the channel 16 that includes the aluminum material 24 to gain access to the aluminum material 24. As illustrated in FIG. 12, aluminum material 24 is wet etched away, leaving channels 16 for microfluidic communication. Next, FIG.
Angular evaporation or angular deposition (angular dep
of the oxide material 32 or any other type of material that does not react with DNA is grown or deposited on the surface of the oxide material 26. This angular deposition or evaporation technique allows the voids 30 to be filled without any oxide material 32 reaching the channels 16 formed in the oxide layer 26.

In a preferred embodiment, holding well 14 supplies fluid to dip well 12 through microfluidic channel 16. To achieve such fluid delivery through the channel 16, capillary action can be utilized for fluid delivery and reduced pressure can be used in the wells for fluid delivery from the holding well to the dip well. It is possible, or disclosed that pressurization to the holding well is used.

Referring now to FIG. 14, schematically illustrated in a side cross-sectional view is a portion of a fluid storage chip 10 having a plurality of retention wells 14 and dip wells 1.
2 places the holding well 14 and the dip well 12 in fluid communication,
Shown are a plurality of microfluidic channels 16 formed therebetween. In this particular embodiment, the channels 16 are illustrated as being formed at multiple planar levels within the silicon wafer 20. With this plurality of levels, the fluid reservoir 1
The overall size of 0 is smaller. In the preferred embodiment, the overall dimensions of the preferred embodiment fluid reservoir 10 are about 2 mm square. The dip well 12 is located at the center, which is about 200 μm square. Thus, if multiple planar heights are utilized to create the channels 16, dimensions less than 2 mm square may be provided to the fluid reservoir 10.

During use of the bio-array fabrication system, in addition to the fluid reservoir 10, the tip 2
A dimensional array is formed of silicon material of the desired size and shape using selective etching techniques. Such as the type of sensing solution typically used in DNA research / diagnosis,
When the tip is immersed in the liquid held in the dip well 12, the tip holds a small amount of liquid sample due to surface tension. Further information on the use of stamps and bio-array fabrication systems, entitled "Bio-Array Fabrication Systems and Methods for Controlled Delivery of Liquids", has Patent Attorney Specification No. CR-99-030,
Co-pending patent application filed at the same time as this specification, assigned to the same successor, and incorporated herein by reference, and "having a silicon tip for controlled delivery of liquids" Stamp and method of creation ", having patent attorney docket number CR-99-029, filed concurrently with this specification, assigned to the same successor, and incorporated herein by reference. Both are found in pending patent applications.

Thus, a fluid reservoir or storage chip comprising a plurality of retention wells, dip wells and microfluidic channels is disclosed for use in bio-array fabrication systems sought for controlled delivery of liquids. . As disclosed, this fluid reservoir utilizes well-known silicon process technology. Fluid reservoirs are expected to be used in the field of protein and DNA hybridization array formation. Accordingly, such examples are intended to be encompassed by this disclosure.

Further preferred embodiments are shown in Figures 15-19. In particular, illustrated in FIG. 15 is a side view illustrating a portion of a large array fabrication system, and further illustrated is a stamp, generally designated 10, as part thereof. It includes a plurality of tips 12. As illustrated, the stamp 10 includes a plurality of spatially separated tips 12 formed utilizing semiconductor industry technology. In particular, in this particular embodiment, the tip 12 is formed using isotropic and anisotropic etching techniques (currently under consideration). Stamp 10 including tip 12 is preferably formed of a silicon material. During fabrication, the tip 12 is deposited with a thick oxide or nitride layer and a mask such as photoresist, predetermined to allow the formation of the tip 12 with different but controlled tip edges and shapes. It is formed by subjecting to wet and dry etching under the above conditions. Tip 12
As currently considered, when immersed in a fluid of constant viscosity, they can retain a small amount of liquid, especially sensing liquid, as a result of surface tension (illustrated in Figure 5).

Referring now to FIGS. 16-19, illustrated are the various steps required to make a stamp having a silicon tip for controlled delivery of liquid. In particular, illustrated is a portion of stamp 10, illustrating the creation of a single tip 12 included as part of a plurality of tips 12 formed as part of stamp 10. As previously mentioned, a two-dimensional array of tips 12 is formed using the selective etching technique in a silicon material of the required size and shape. The tip 12 retains a small sample due to surface tension when immersed in a liquid, such as a sensing liquid typically of the type utilized in DNA research / diagnosis.

Referring again to FIGS. 16-19, illustrated in an enlarged side cross-sectional view is a portion of stamp 10, illustrating the creation of a single tip 12. As illustrated,
The tip 12 is formed using isotropic and anisotropic etching techniques. Stamp 1
The 0 is formed by a base 14 having a tip 12 protruding therefrom (illustrated in FIG. 15). As illustrated in FIG. 16, the first stage of creating the stamp 10 is
Providing the silicon material that forms the base 14. The base 14 includes a silicon material 16 upon which a top surface 17, silicon dioxide 18 and a mask 20, such as photoresist, are formed.

Referring now to FIG. 17, the silicon dioxide 18 is selectively etched down to the silicon material 16 and the photoresist or mask 20 is removed following the selective etching, as shown. As illustrated in FIG. 18, isotropic etching is used to form the angled portion 22, thus forming a pointed structure, and as illustrated in FIG. 19, anisotropic etching is used. It forms a vertical side 24, thus providing a depth stop (currently under consideration) and a narrower pitch between the tips 12.
This array of selective etching techniques results in well-defined shapes and sizes of tips 12 using traditional materials and facilitates integration with other electronics. In a preferred embodiment, the tip 12 is 5 μm-1 mm, preferably 30 μm or less,
Space at a pitch in the range. With this pitch interval, 30 at 1x1 cm,
000-50,000 tips 12 will be placed.

In this particular embodiment, each tip 12 has a side surface 24 that is substantially perpendicular to the base 14.
, And an angled portion 22 to a point defining an end point 28 of the tip 12. The tip 12 is formed so that the detection liquid can be accurately attached. In another embodiment, the silicon dioxide 18 remains at the end points 28, thus providing a larger area for the sensing liquid to adhere. Although silicon dioxide 18 is specifically mentioned, it should be understood that other oxides, nitrides or metals can be used. This material, which remains at the end point 28, allows for the definition of hydrophilic and hydrophobic moieties or regions suitable for use with certain liquids. For example, the end point 2 of the tip 12
In order to better control the amount of DNA-containing liquid that attaches to the horns 8, the angled portions 22, sides 24 and surface 30 can be treated to obtain hydrophobic portions.

In this particular embodiment, as illustrated in FIG. 19, a tip having a particular height “h” measured from surface 30 of base 14 to end point 28 of tip 12 during the fabrication process. 12 is formed. This particular depth dimension provides a stopping point when the stamp is submerged in the sensing liquid typically contained in a complementary formed well structure called a dip well. As illustrated in FIG. 19, the sensing liquid 32 adheres to the end point 28 of the tip 12 due to surface tension. With this depth dimension,
A precise “dip” of the end point 28 of the tip 12 into the sensing liquid 32 is provided. It should be understood that the remaining silicon dioxide 18 has been removed from the end points 28, as illustrated.

Alternatively, as illustrated in FIG. 15, the surface 26 of the stamp 10 provides a stopping point when the stamp 10 is immersed in a sensing liquid typically contained in a complementary formed well structure. In particular, the surface 26 of the stamp 10 contacts the surface of a fluid storage chip containing a plurality of dip wells so that the end point 28 is accurately placed in the sensing liquid.

During operation, the stamp 10 is dipped into a complementarily formed fluid reservoir, in particular a dip well, for contact of the surface 26 of the stamp 10 and the surface of the storage tip or other similar depth stop. Take the right position for. Illustrated in the figure is a particular type of stopping method that allows for accurate positioning of the stamp 10 relative to the storage chip, but with respect to the storage chip, such as a protrusion from the stamp 10 that rests on a recessed portion of the storage chip. It should be understood that other means for placing the stamp 10 are envisioned in this disclosure. Next, depending on the size and shape of the tip 12, the sensing liquid 32 is attached to the end point 28 of the tip 12. Once the specified amount of the detection liquid 32 has adhered to the tip 12, the stamp 10 is placed on the sensor plate (not shown). The sensor plate, once stamped, provides multiple test sites. It should be appreciated that multiple stamping acts can be utilized to try to increase the density of test sites on the surface of the sensor plate. Accordingly, a stamp with a silicon tip for controlled delivery of a liquid and a method of making the stamp for use in a bio-array fabrication system are disclosed. As disclosed, this system utilizes well-known silicon process technology. Stamps are protein and D
It is envisioned for use in the field of large array formation for large molecule adhesion events such as NA hybridization array formation. Accordingly, such examples are intended to be encompassed by this disclosure.

A further preferred embodiment is shown in Figures 20-25. Figures 20-24 show bio-
1 illustrates a plurality of components that make up an array fabrication system and use of the bio-array fabrication system for controlled delivery of liquids according to the present invention. In particular, FIG.
Illustrated in FIG. 1 is generally designated as 10, stamp 12 and fluid reservoir 1
FIG. 4 is a side view illustrating a portion of a bio-array fabrication system including # 4. Stamp 12 includes a plurality of tips 16 (currently under consideration). The fluid reservoir 14 has a plurality of dip wells 18 (formed therein) to "hold" the sensing liquid 20.
(Currently being considered) and multiple retention wells (not shown). It is understood that the dip well 18 is formed in the fluid reservoir 14 and is “pumped” with the sensing liquid through a plurality of microfluidic channels (not shown) connecting the dip well 18 and the holding well. Should.

As illustrated, the stamp 12 includes a plurality of spaced apart tips 16 formed utilizing semiconductor manufacturing techniques. In particular, in this particular embodiment, the tip 16 is formed utilizing isotropic and anisotropic etching techniques. Stamp 12 and, in addition, tip 16 are formed of silicon. During fabrication, the tip 16 is deposited with an oxide or nitride mask, wet and under predetermined conditions to allow the formation of the tip 16 with different but controlled tip edges and shapes. It is formed by subjecting it to dry etching. The tip 16 can retain a small amount of liquid, particularly the sensing liquid 20, as a result of surface tension when immersed in a constant viscous fluid, as currently considered.

Generally, the two-dimensional array of tips 16 is formed of silicon of the desired size and shape using selective etching techniques. The tip 16 retains a small liquid sample due to surface tension when immersed in a liquid, such as a sensing liquid 20 of the type typically used in DNA research / diagnosis.

Referring now to the fluid reservoir 14, the fluid reservoir 14 described above is formed including a plurality of dip wells 18 and a plurality of holding wells (not shown). The dip well 18 is formed to hold a sensing liquid 20 and is pumped with the sensing liquid through a plurality of microfluidic channels 22 (currently under consideration). During use, the tip 16 is dipped into a dip well 18 that holds a sensing liquid 20 for use. In order to obtain different sensing or sample liquids on different tips 16, the dip well 18 in which each tip is dipped must contain the correct liquid 20. This is accomplished by forming an array of small wells that can hold different liquids.
Fluid reservoir 14 as illustrated by the spacing between tip 16 and dip well 18.
Are formed to be complementary to the stamp 12.

Referring now to FIG. 21, illustrated in a simplified diagram is stamp 1
2 and the sensing area or sensor plate 30. The sensor plate 30 is defined as the material on which the bio-array pattern is formed. During production of the bio-array sensor plate, a stamp 12 having thousands of tips 16 formed therein is aligned with a reservoir 14 and picks up a sensing liquid as described above, after which the stamp 12 is a sensor. Aligned to plate 30 and simultaneously transferred thousands of different sensing liquids to sensor plate 30 in one stamping step.

Referring now to FIG. 22, stamp 12 illustrates in an enlarged side cross-sectional view.
And includes the tip 16. As illustrated, the stamp 16 has a tip 16
Is formed using isotropic and anisotropic etching techniques. The stamp 12 is further formed with a base 32 having a tip 16 protruding therefrom. During fabrication, the tip 16 is formed with a particular height "h" measured from the surface 34 of the base 32 to the end point of the tip 16 as illustrated. As illustrated in FIG. 3, the sensing liquid 20 adheres to the end point 36 of the tip 16 due to surface tension.

In this particular embodiment, each tip 16 has a side surface 38 that is substantially perpendicular to the base 32.
, And with an angled portion 40 to a point defining the end point 36 of the tip 16. The tip 16 is formed so that the detection liquid 20 can be accurately attached.

Referring now to FIGS. 23 and 24, illustrated are side cross-sectional views of a portion of fluid reservoir 14 showing formation of dip well 18, and dip well 1.
8 is a plan view of a fluid reservoir 14 illustrating an arrangement of 8 and a plurality of microfluidic channels 22 (currently under consideration). Fluid reservoir 14 is formed using standard semiconductor processing techniques. In particular, as illustrated in FIG. 24, the fluid reservoir 14 is formed to include a plurality of dip wells 18, generally similar to forming a semiconductor chip that includes a plurality of fanned bond pads. In particular, a plurality of microfluidic channels 22 are formed so as to be in fluid communication with a large fluid reservoir 50 or holding well formed around reservoir 14. Retention wells 50 are typically on the order of 100 microns, while actual array dimensions can be on the order of tens of microns.

As illustrated in FIG. 24, the fluid reservoir 14, and in particular the dip well 18, is formed with a depth “h”. The depth “h” is the depth “h” of the stamp 12 as described above.
is complementary to "h". This same depth dimension allows the end point 36 of the tip 16 to be accurately “immersed” in the sensing liquid 20. Further, the surface 52 of the fluid reservoir 14
It provides a stopping point when the stamp 12 is dipped into the dip well 18. In particular,
Contact of the surface 34 of the stamp 12 with the surface 52 of the fluid reservoir 14 allows for accurate placement of the end point 36 in the sensing liquid 20. As illustrated, the detection liquid 2
0 is pumped into the dip well 18 through the microfluidic channel 22. In this particular embodiment, holding wells 50 can serve for a single dip well 18 or multiple dip wells 18, and each dip well 18 holds a different sensing solution 20, depending on the particular application of the device. It should be understood that this disclosure anticipates that a greater number of retention wells 50 can be provided, as possible.

In operation, the stamp 12 is dipped into a reservoir 14, in particular a dip well 18,
Accurate placement due to contact between surface 34 of stamp 12 and surface 52 of reservoir 14. Although illustrated in the figure is a particular type of stopping method that allows the stamp 12 to be accurately positioned relative to the reservoir 14, such as a fluid reservoir, such as a protrusion from the stamp 12 that rests on a recessed portion of the reservoir 14. It should be understood that other means for placing the stamp 12 relative to 14 are envisioned in this disclosure. Next, the detection liquid 20 is added to each tip 1
Attach to 6. Once the specified amount of the detection liquid 20 has adhered to the tip 16, the stamp 1
Place 2 against sensor plate 30. As illustrated in FIG. 6, the sensor plate 30, once stamped, provides multiple test sites 60. It should be appreciated that multiple stamping actions can be utilized to try to densify the test sites 60 on the surface 61 of the sensor plate 30.

Thus, a system for bio-array fabrication and a method of delivering a controlled amount of liquid onto a surface to form a bio-array are disclosed. As disclosed, this system utilizes well-known silicon process technology. This system is a protein,
It is expected to be used in RNA and DNA hybridization array formation. Accordingly, such examples are intended to be encompassed by this disclosure.

[0080]   All cited references are incorporated herein by reference.

[Brief description of drawings]

FIG. 1 is a plan view of a fluid reservoir.

FIG. 2 is a simplified side view of the steps utilized during fabrication of a fluid reservoir.

FIG. 3 is a simplified side view of the steps utilized during fabrication of a fluid reservoir.

FIG. 4 is a simplified side view of the steps utilized during fabrication of a fluid reservoir.

FIG. 5 is a simplified plan view of the stages utilized during fabrication of the fluid reservoir.

FIG. 6 is a simplified plan view of the stages utilized during fabrication of the fluid reservoir.

FIG. 7 is a simplified plan view of the stages utilized during fabrication of the fluid reservoir.

FIG. 8 is a simplified plan view of the stages utilized during fabrication of the fluid reservoir.

FIG. 9 is a simplified plan view of the stages utilized during fabrication of the fluid reservoir.

FIG. 10 is a simplified plan view of the stages utilized during fabrication of the fluid reservoir.

FIG. 11 is a simplified side view of the steps utilized during the creation of channel voids.

FIG. 12 is a simplified side view of the steps utilized during the creation of channel voids.

FIG. 13 is a simplified side view of the steps utilized during the creation of channel voids.

FIG. 14 is a simplified cross-sectional view of a multi-layer fluid storage chip.

FIG. 15 is a simplified side view of a stamp including multiple tips.

FIG. 16 is a simplified side view of the steps used during production of a stamp including multiple tips.

FIG. 17 is a simplified side view of the steps used during production of a stamp including multiple tips.

FIG. 18 is a simplified side view of the steps used during production of a stamp including multiple tips.

FIG. 19 is a simplified side view of the steps used during production of a stamp including multiple tips.

FIG. 20 is a simplified side view of a bio-array fabrication system stamp and reservoir.

FIG. 21 is a simplified side view of the stamp and sensor plate area of the bio-array fabrication system.

FIG. 22 is an enlarged side view of a plurality of tips forming a stamp of a bio-array fabrication system.

FIG. 23 is an enlarged side view of a dip well formed in a fluid reservoir of a bio-array fabrication system.

FIG. 24 is a plan view of a fluid reservoir.

FIG. 25 is a plan view of a sensor plate.

FIG. 26 shows a “one element” system.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 37/00 102 C12N 15/00 F (31) Priority claim number 09/466, 314 (32) Priority date December 17, 1999 (December 17, 1999) (33) Priority claiming countries United States (US) (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ , T M), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT , LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor George N Maracas United States 85048-9512 East Bighorn Avenue 2613 (Phoenix, Arizona) 72) Inventor Kumar Siraragi United States 85226 961, North Florence Drive, Chandler, Ariz. John Tressec, Jr. United States 85048 Apartment 3027, Lakewood Parkway East, 3830 F-Term (Reference) 4B024 AA11 CA09, Phenix, Arizona HA14 HA19 4B029 AA21 AA23 BB20 CC03

Claims (7)

[Claims] 1. A) at least one holding well; b) at least one dip well; and c) at least one microflow connecting the holding well and the dip well     Body channel, An apparatus including a fluid reservoir including a first substrate including. 2. The fluid reservoir comprises: a) multiple retention wells; b) multiple dip wells; and c) a plurality of microfluids, each connecting a holding well and a dip well     channel, The device of claim 1, comprising: 3. The apparatus of claim 1 or claim 2, further comprising a stamp that includes a second substrate that includes a plurality of donor tips. 4. The apparatus of claim 1, claim 2 or claim 3, wherein the substrate comprises a semiconductor material. 5. A substrate comprising first and second surfaces, said first surface comprising a plurality of storage wells, said second surface comprising a plurality of donor heads, and said substrate further comprising: A dispenser for dispensing liquid, comprising a plurality of microfluidic channels each connecting a storage well to a dispensing head. 6. A fluid reservoir comprising: a) i) a plurality of holding wells; ii) a plurality of dip wells; and iii) a plurality of microfluidic channels, each connecting the holding well and the dip well. Providing, but the holding wells, the dipwells and the microfluidic channels contain a fluid containing a biological reagent; b) a first substrate containing a plurality of donor tips. Providing a stamp comprising; c) contacting the donor tip group into the dip well group and loading the tip group with the biological agent; and d) contacting the tip group to a sensor plate. To form a bioarray,
A method of making a bioarray, including: 7. A dispenser comprising a substrate comprising first and second surfaces, the first surface comprising a plurality of storage wells and the second surface comprising a plurality of dispensing heads. And the substrate further comprises a plurality of microfluidic channels each connecting a storage well to a donor head, and the storage well, the donor head and the channel containing a fluid containing a biological reagent. And b) contacting the donor head with a sensor plate to form a bioarray.