Method of mixing by intermittent centrifugal force Number:7,147,362 from the United States Patent and Trademark Office (PTO) owispatent An apparatus for mixing fluids comprises a housing having at least a portion of its interior, for example, a channel, with capillary dimensions and at least one structural member in the interior adjacent an end of the channel. The dimensions of and placement of the structural member are sufficient such that intermittent application of centrifugal force to the interior of the housing causes movement of the fluid therein. The housing may comprise a mixing chamber or area that is in fluid communication with the interior. The apparatus may comprise a mechanism adapted to generate centrifugal force within the housing to cause movement of fluid in the interior of the housing without exit of fluid therefrom.<H intermittent, centrifugal, Method, of, mixin, 7147362.html, Patent, pto, 7147362

Title: Method of mixing by intermittent centrifugal force

Abstract: An apparatus for mixing fluids comprises a housing having at least a portion of its interior, for example, a channel, with capillary dimensions and at least one structural member in the interior adjacent an end of the channel. The dimensions of and placement of the structural member are sufficient such that intermittent application of centrifugal force to the interior of the housing causes movement of the fluid therein. The housing may comprise a mixing chamber or area that is in fluid communication with the interior. The apparatus may comprise a mechanism adapted to generate centrifugal force within the housing to cause movement of fluid in the interior of the housing without exit of fluid therefrom.

Patent Number: 7,147,362 Issued on 12/12/2006 to Caren, et al.

Inventors: Caren; Michael P. (Palo Alto, CA), Schembri; Carol T. (San Mate, CA)
Assignee: Agilent Technologies, Inc. (Santa Clara, CA)

Appl. No.: 10/687,276

Filed: October 15, 2003

Current U.S. Class: 366/135 ; 366/150.1; 366/228; 366/235; 366/DIG.1; 366/DIG.3; 422/209; 435/287.2; 435/303.3; 435/6

Current International Class: B01F 5/06 (20060101)

Field of Search: 366/131,135,150.1,213,214,220,225,228,235,DIG.1,DIG.3 435/4,6,287.2,303.3 422/72,209

References Cited [Referenced By]

U.S. Patent Documents


3744975	July 1973	Mailen
4426451	January 1984	Columbus
5104813	April 1992	Besemer et al.
5230866	July 1993	Shartle et al.
5693233	December 1997	Schembri
5804384	September 1998	Muller et al.
5837832	November 1998	Chee et al.
5912134	June 1999	Shartle
6103199	August 2000	Bjornson et al.
6296020	October 2001	McNeely et al.
6319469	November 2001	Mian et al.
6395232	May 2002	McBride
6448090	September 2002	McBride
6451188	September 2002	Sundberg et al.
6458599	October 2002	Huang
6488827	December 2002	Shartle
6521182	February 2003	Shartle et al.
6527432	March 2003	Kellogg et al.
2002/0097632	July 2002	Kellogg et al.
2002/0177159	November 2002	Bedilion et al.
2005/0084866	April 2005	Caren et al.

Foreign Patent Documents


WO 99/42605	Aug., 1999	WO

Primary Examiner: Sorkin; David

Claims

What is claimed is:

1. A method for mixing a fluid, said method comprising: (a) introducing a fluid into a housing of an apparatus, said apparatus comprising: (i) a housing having an interior comprising an interior channel with capillary dimensions, said interior channel comprising a plurality of biopolymer features arranged in a predetermined manner on an interior surface thereof, (ii) an opening at a proximal end of said interior channel, (iii) a chamber at a distal end of said interior channel, (iv) a structural member in said interior channel adjacent said distal end, the dimensions of and placement of said structural member being sufficient such that intermittent application of centrifugal force to said interior channel causes movement of said fluid between said interior channel and said chamber, and (v) a mechanism that intermittently generates centrifugal force on the interior of said housing to cause movement of said fluid in said channel, and (b) generating intermittent centrifugal force to cause repetitive movement of said fluid between said interior channel and said chamber sufficient to cause mixing of said fluid by agitation.

2. A method according to claim 1 wherein said intermittent centrifugal force is generated by rotating said mechanism.

3. A method according to claim 1 wherein said interior channel comprises a linear array of biopolymer features.

4. A method according to claim 3 wherein said linear array is a linear microarray.

5. A method according to claim 4 wherein said linear microarray comprises more than one thousand features.

6. A method according to claim 1 wherein said biopolymer features are polynucleotides or polypeptides.

7. A method according to claim 1 wherein said method is a method for conducting hybridization reactions.

8. A method for conducting chemical reactions, said method comprising: (a) introducing a fluid sample into a housing of an apparatus, said apparatus comprising: (i) a housing having an interior comprising an interior channel with capillary dimensions, said interior channel comprising a linear array of biopolymer features arranged in a predetermined manner on an interior surface thereof, (ii) an opening at a proximal end of said interior channel, (iii) a chamber at a distal end of said interior channel, (iv) a structural member in said interior channel adjacent said distal end, the dimensions of and placement of said structural member being sufficient such that intermittent application of centrifugal force to said interior channel causes movement of said fluid between said interior channel and said chamber, and (v) a mechanism that intermittently generates centrifugal force on the interior of said housing to cause movement of said fluid in said channel, and (b) incubating said fluid sample in said housing under conditions for carrying out said chemical reactions and during said incubation generating intermittent centrifugal force to cause repetitive movement of said fluid sample between said linear array and said chamber sufficient to cause mixing of said fluid sample by agitation.

9. A method according to claim 8 wherein said intermittent centrifugal force is generated by rotating said housing.

10. A method according to claim 8 wherein said linear array is a linear microarray.

11. A method according to claim 10 wherein said linear microarray comprises more than one thousand features.

12. A method according to claim 8 wherein said biopolymer features are polynucleotides or polypeptides.

13. A method according to claim 8 wherein said method is a method for conducting hybridization reactions.

14. A method for conducting hybridization reactions, said method comprising: (a) introducing a fluid sample into an opening at a proximal end of a housing comprising a linear microarray of biopolymer features for hybridizing to analytes in said sample, said housing having an interior with internal capillary dimensions, a mixing area separate from said linear array at a distal end of said housing and a structural member in said housing adjacent said distal end, the dimensions of and placement of said structural member being sufficient such that intermittent application of centrifugal force to said housing causes motion of said fluid therein, and (b) incubating said fluid sample in said housing under conditions for carrying out said hybridization reactions and during said incubation generating intermittent centrifugal force to cause repetitive reciprocal movement of said fluid sample between said linear array and said mixing area such that said fluid sample is mixed by agitation.

15. A method according to claim 14 wherein said intermittent centrifugal force is generated by rotating said housing.

16. A method according to claim 14 further comprising, subsequent to said incubation, increasing said centrifugal force sufficient to cause said fluid to exit said interior.

17. A method according to claim 16 further comprising introducing a wash fluid into said housing and generating intermittent centrifugal force sufficient to cause agitation of said wash fluid but insufficient to cause said wash fluid to exit said housing.

18. A method according to claim 17 further comprising increasing said centrifugal force sufficient to cause said fluid to exit said interior.

19. A method according to claim 14 further comprising examining said linear array for the results of said hybridization reactions.

20. A method according to claim 19 comprising forwarding data representing a result obtained from said examining.

21. A method according to claim 20 wherein the data is transmitted to a remote location.

22. A method according to claim 19 comprising receiving data representing a result of an interrogation obtained by said examining.

23. A method according to claim 14 wherein said housing is part of a microfluidic system.

24. A method according to claim 14 wherein said housing is a channel in a microfluidic system.

25. A method according to claim 14 wherein said features are polynucleotides or polypeptides.

26. A method according to claim 14 wherein said linear microarray comprises more than one thousand features.

Description

BACKGROUND OF THE INVENTION

The present invention relates to apparatus and methods for conducting chemical and biological analyses using linear arrays. More particularly, the invention relates to apparatus and methods for carrying out mixing operations in hybridization reactions using linear microarrays. The invention has utility in fields relating to biology, chemistry and biochemistry.

Determining the nucleotide sequences and expression levels of nucleic acids (DNA and RNA) is critical to understanding the function and control of genes and their relationship, for example, to disease discovery and disease management. Analysis of genetic information plays a crucial role in biological experimentation. This has become especially true with regard to studies directed at understanding the fundamental genetic and environmental factors associated with disease and the effects of potential therapeutic agents on the cell. Such a determination permits the early detection of infectious organisms such as bacteria, viruses, etc.; genetic diseases such as sickle cell anemia; and various cancers. New methods of diagnosis of diseases, such as AIDS, cancer, sickle cell anemia, cystic fibrosis, diabetes, muscular dystrophy, and the like, rely on the detection of mutations present in certain nucleotide sequences. This paradigm shift has lead to an increasing need within the life science industries for more sensitive, more accurate and higher-throughput technologies for performing analysis on genetic material obtained from a variety of biological sources.

Unique or misexpressed nucleotide sequences in a polynucleotide can be detected by hybridization with a nucleotide multimer, or oligonucleotide, probe. Hybridization reactions between surface-bound probes and target molecules in solution may be used to detect the presence of particular biopolymers. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules. These techniques rely upon the inherent ability of nucleic acids to form duplexes via hydrogen bonding according to Watson-Crick base-pairing rules. The ability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research. An oligonucleotide probe employed in the detection is selected with a nucleotide sequence complementary, usually exactly complementary, to the nucleotide sequence in the target nucleic acid. Following hybridization of the probe with the target nucleic acid, any oligonucleotide probe/nucleic acid hybrids that have formed are typically separated from unhybridized probe. The amount of oligonucleotide probe in either of the two separated media is then tested to provide a qualitative or quantitative measurement of the amount of target nucleic acid originally present.

Such reactions form the basis for many of the methods and devices used in the field of genomics to probe nucleic acid sequences for novel genes, gene fragments, gene variants and mutations. The ability to clone and synthesize nucleotide sequences has led to the development of a number of techniques for disease diagnosis and genetic analysis. Genetic analysis, including correlation of genotypes and phenotypes, contributes to the information necessary for elucidating metabolic pathways, for understanding biological functions, and for revealing changes in genes that confer disease. Many of these techniques generally involve hybridization between a target nucleotide sequence and a complementary probe, offering a convenient and reliable means for the isolation, identification, and analysis of nucleotides. The surface-bound probes may be oligonucleotides, peptides, polypeptides, proteins, antibodies or other molecules capable of reacting with target molecules in solution.

Direct detection of labeled target nucleic acid hybridized to surface-bound polynucleotide probes is particularly advantageous if the surface contains a mosaic of different probes that are individually localized to discrete, known areas of the surface. Such ordered arrays of probes are commonly referred to as "biochip" arrays. Biochip arrays containing a large number of oligonucleotide probes have been developed as tools for high throughput analyses of genotype and gene expression. Oligonucleotides synthesized on a solid support recognize uniquely complementary nucleic acids by hybridization, and arrays can be designed to define specific target sequences, analyze gene expression patterns or identify specific allelic variations.

In one approach, cell matter is lysed, to release its DNA as fragments, which are then separated out by electrophoresis or other means, and then tagged with a fluorescent or other label. The resulting DNA mix is exposed to an array of oligonucleotide probes, whereupon selective attachment to matching probe sites takes place. The array is then washed and imaged so as to reveal for analysis and interpretation the sites where attachment occurred.

One typical method involves hybridization with probe nucleotide sequences immobilized in an array on a substrate having a surface area of typically less than a few square centimeters. The substrate may be glass, fused silica, silicon, plastic or other material; typically, it is a glass slide, which has been treated to facilitate attachment of the probes. The mobile phase, containing reactants that react with the attached probes, is placed in contact with the substrate, covered with another slide, and placed in an environmentally controlled chamber such as an incubator. Normally, the reactant targets in the mobile phase diffuse through the liquid to the interface where the complementary probes are immobilized, and a reaction, such as a hybridization reaction, then occurs. The mobile phase targets may be labeled with a detectable tag, such as a fluorescent tag, or chemiluminescent tag, or radioactive label, so that the reaction can be detected. The location of the signal in the array provides the target identification. The hybridization reaction typically takes place over a time period of seconds up to many hours.

Biochip arrays have become an increasingly important tool in the biotechnology industry and related fields. These binding agent arrays, in which a plurality of binding agents are synthesized on or deposited onto a substrate in the form of an array or pattern, find use in a variety of applications, including gene expression analysis, drug screening, nucleic acid sequencing, mutation analysis, and the like. Substrate-bound biopolymer arrays, particularly oligonucleotide, DNA and RNA arrays, may be used in screening studies for determination of binding affinity and in diagnostic applications, e.g., to detect the presence of a nucleic acid containing a specific, known oligonucleotide sequence.

The pattern of binding by target molecules to biopolymer probe spots on the biochip forms a pattern on the surface of the biochip and provides desired information about the sample. Hybridization patterns on biochip arrays are typically read by optical means, although other methods may also be used. For example, laser light in the Agilent Technologies Inc. GeneArray Scanner excites fluorescent molecules incorporated into the nucleic acid probes on a biochip, generating a signal only in those spots on the biochip that have a target molecule bound to a probe molecule, thus generating an optical hybridization pattern. This pattern may be digitally scanned for computer analysis. Such patterns can be used to generate data for biological assays such as the identification of drug targets, single-nucleotide polymorphism mapping, monitoring samples from patients to track their response to treatment, and assess the efficacy of new treatments.

One type of linear array is a one-dimensional array of features bound in a non-diffusive manner to a surface, which may be located on the inside of an enclosed microchannel. The order of the features identifies each feature, which allows selective identification of target molecules. One such linear array is disclosed in U.S. Pat. No. 5,804,384 (Muller, et al.). The devices of Muller, et al., consist of a tube containing a linear array of specific binding elements that each have capture probes specific for a target analyte.

Inadequate mixing is a particular problem in chemical and biological assays where very small samples of chemical, biochemical, or biological fluids are typically involved. Inhomogeneous solutions resulting from inadequate mixing can lead to poor hybridization kinetics, low efficiency, low sensitivity, and low yield. With inadequate mixing, diffusion becomes the only means of transporting the reactants in the mobile phase to the interface or surface containing the immobilized reactants. In such a case, the mobile phase can become depleted of reactants near the substrate as mobile molecules become bound to the immobile phase.

Methods for mixing relatively large volumes of fluids may utilize conventional mixing devices that mix the fluids by shaking the container, by a rapid mechanical up and down motion, or by the use of a rocking motion that tilts the container filled with the fluids in a back and forth motion. The conventional mixing methods normally cannot be utilized for small volumes of fluid such as thin films of fluids in capillary chambers because the capillary strength of the containment system often significantly exceeds the forces generated by shaking or rocking, thereby preventing or minimizing fluid motion in the film. This is because most or all of the fluid is so close to the walls of the chamber that there is virtually no bulk phase so that surface interactions predominate.

There remains a need in the art for efficient and effective methods and apparatus for mixing fluids in small chambers such as capillary chambers in which linear arrays are housed.

SUMMARY OF THE INVENTION

One embodiment of the present invention is an apparatus for mixing fluids. The apparatus comprises a housing having at least a portion of its interior, for example, a channel, with capillary dimensions and at least one structural member in the interior adjacent an end of the channel. The dimensions of and placement of the structural member are sufficient such that intermittent application of centrifugal force to the interior of the housing causes movement of the fluid therein. The housing may comprise a mixing chamber or area that is in fluid communication with the interior. The apparatus may comprise a mechanism for generating centrifugal force, e.g. a rotatable support for rotating the housing to produce the centrifugal force. The mechanism is adapted to produce intermittent centrifugal force to cause movement of fluid in the interior of the housing without exit of fluid therefrom. The apparatus may optionally comprise a fluid dispensing device. The interior of the apparatus may comprise a linear array of features for conducting chemical reactions and the apparatus may be employed to conduct such reactions.

Another embodiment of the present invention is an apparatus for conducting hybridization reactions. The apparatus comprises a housing having an interior with capillary dimensions, a structural member in the interior adjacent an end of the housing, and a mechanism adapted to generate centrifugal force in the interior of the housing. The interior comprises a linear microarray of biopolymers for conducting hybridization reactions. The interior may also comprise a mixing area adjacent said linear microarray. The dimensions of and placement of the structural member are sufficient such that intermittent application of centrifugal force to the interior causes reciprocal movement of the fluid in the interior between said linear microarray and said mixing area. Optionally, the apparatus may comprise a fluid dispensing device.

Another embodiment of the present invention is a method for mixing a fluid. The method comprises introducing a fluid into a housing of an apparatus as described above and generating intermittent centrifugal force to cause movement of the fluid but insufficient to cause the fluid to exit the housing.

Another embodiment of the present invention is a method for conducting chemical reactions. The method comprises introducing a sample into a housing of an apparatus as described above and incubating the sample in the housing under conditions for carrying out the chemical reactions. During the incubation intermittent centrifugal force is generated to cause reciprocal movement of the fluid in the interior of the housing between the linear array and the mixing area but insufficient to cause the fluid to exit the housing.

Another embodiment of the present invention is a method for conducting hybridization reactions. A sample is introduced into a housing comprising a linear microarray of features for hybridizing to analytes in the sample and a mixing area. The housing has internal capillary dimensions and a structural member in the interior adjacent an end of the housing. The dimensions of and placement of the structural member are sufficient such that intermittent application of centrifugal force to the interior causes motion of the fluid in the housing. The sample is incubated in the housing under conditions for carrying out the hybridization reactions. During the incubation intermittent centrifugal force is generated to cause reciprocal movement of the fluid between the linear array and the mixing area but insufficient to cause the fluid to exit the housing. Rotating the housing may generate the intermittent centrifugal force. Optionally, the centrifugal force may be increased to a level sufficient to cause the fluid to exit the interior. A wash fluid may be introduced into the housing and intermittent centrifugal force may be generated sufficient to cause movement of the wash fluid but insufficient to cause the wash fluid to exit the housing. Optionally, the centrifugal force may be increased to a level sufficient to cause the fluid to exit the interior.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures are included to better illustrate the embodiments of the devices and techniques of the present invention. The figures are not to scale and some parts of the figures may be exaggerated for the purpose of illustrating certain aspects or embodiments of the present invention.

FIG. 1 is a perspective view taken from the top of a portion of an embodiment of an apparatus in accordance with the present invention.

FIG. 2 is a perspective view taken from the top of a larger portion of the embodiment of FIG. 1.

FIG. 3 is a perspective view taken from the top of a portion of another embodiment of an apparatus in accordance with the present invention.

FIG. 4 is a perspective view taken from the top of a portion of another embodiment of an apparatus in accordance with the present invention.

FIG. 5 is a perspective view taken from the side of an embodiment of an apparatus for rotating a plurality of linear arrays.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

The present invention utilizes a capillary valve and centrifugal force to induce mixing in fluids. In one embodiment the capillary valve may comprise small structural members, which essentially act as a capillary leash, i.e., a way of controlling the movement of fluid in a capillary environment. Centrifugal force may be employed to move fluid in a capillary channel within a housing while retaining the fluid within the channel or to move the fluid to a mixing area and back to the channel. To remove fluid from the channel, the centrifugal force is increased to a level to overcome the capillary forces within the channel. The centrifugal force may be applied to the channel by rotating the channel.

The present invention provides a multi-function valve that acts as a leash to control movement of the fluid within a capillary environment and to control exit of fluid from the capillary environment. The valve may be fabricated at the same time as the fabrication of the capillary housing. The functions of the valve may be controlled by the same parameter, i.e., the rotational speed of the capillary housing. A combination of the multi-function valve and centrifugal force may be employed to mix fluids within, and to control exit of fluids from, the capillary housing. The apparatus of the present invention is easy to automate and to fabricate.

The term "mixing" includes mixing of multi-component systems as well as solutions that are inhomogeneous due to depletion of certain components over other components present in a complex mixture of components. For example, a sample comprising a plurality of analytes applied to a linear array experiences localized depletion of certain analytes as the sample contacts the linear array thus resulting in an inhomogeneous solution. Mixing as used herein includes agitation of inhomogeneous solutions or samples to overcome this type of inhomogeneity.

The phrase "adapted to" or "adapted for" is used herein with respect to components of the present apparatus. The components of the present apparatus are adapted to perform a specified function by a combination of hardware and software. This includes the structure of the particular component and may also include a microprocessor, embedded real-time software and I/O interface electronics to control the sequence of operations of the invention.

The housing is any enclosure in which at least a portion of the interior has capillary dimensions. The housing may comprise a microchannel. In one approach, the microchannel is part of a microfluidic system. Microfluidic systems have been developed for performing chemical, clinical, and environmental analysis of chemical and biological specimens. The term microfluidic system refers to a system or device having a network of chambers connected by channels, in which the channels have microscale features, that is, features too small to examine with the unaided eye. The channel often has a capillary dimension, i.e., a cross-sectional area that provides for capillary flow through the channel. At least one of the cross-sectional dimensions, e.g., width, height, diameter, is at least about 1 .mu.m, at least about 10 .mu.m, and is typically no more than about 500 .mu.m, typically no more than about 200 .mu.m. Channels of capillary dimension typically have an inside bore diameter (ID) of from about 1 to about 200 microns, more typically from about 25 to about 100 microns. The term "microfluidic" generally means of or pertaining to fluids and being of a magnitude on the order consistent with capillary dimension. The channel(s) may be part of a microfluidic network or a system of interconnected cavity structures and capillary-size channels configured with a plurality of branches through which fluids may be manipulated and processed. In one simple form, the present apparatus comprise a microchannel in the form of a tube within a housing.

The capillary leash generally comprises one or more structural members in a capillary channel, which may be near an end of a capillary channel. The structural members may be any structure that permits the desired effect of retaining and mixing fluid in the capillary channel at one speed of rotation and releasing the fluid from the capillary channel at another speed of rotation. The structural member may be in or on a wall of the capillary channel or separate therefrom. In general, the structural member changes the capillary dimensions of the capillary in the area of the structure. The change in the capillary dimension may be an increase or a decrease, for example, an increase. The structural member permits fluid in the capillary channel to be maintained in and mixed in the capillary channel at a first predetermined pressure difference across the area of the structure and to break through the area adjacent the structural member when there is a second predetermined pressure difference across the area of the structural member. The structural member may be an indentation or recess in, or protrusion on, a wall of the capillary channel. On the other hand, the structural member may be separate from the wall of the capillary channel and non-movably secured therein. To this end, the separate structural member may be attached to the wall of the capillary channel by means such as adhesives, sonic welding, heat, pressure, spin coating, photolithographic etching and the like. Any adhesive or other means of attachment employed must be compatible with the fluids that will be introduced into the capillary channel during use of the present apparatus. The separate structural member may be secured in the capillary channel by friction fitting and the like. In any event, the separate structural member must be able to withstand the forces applied to the capillary channel and the present device during the course of its use in the methods of the present invention.

The structural member may have any convenient shape such as, for example, circular, oval, rectangular, square, and so forth. Other considerations regarding the structural member relate primarily to capillarity and, thus, pressure and include manufacturability, surface wettability and the like.

The dimensions of the structural member are dependent on the capillary dimensions of the channel comprising the array, the shape of the channel, the length of the channel, the viscosity and/or surface tension of fluid that is to be moved within the capillary housing, the rpm constraints of the motor rotating the linear array, and so forth. For circular capillary channels with interior dimensions of about 10 to about 500 microns, the dimensions of the structural member are about 1 to about 100 microns, about 2 to about 50 microns, about 4 to about 40 microns, about 5 to about 30 microns, about 10 to about 20 microns.

The structural member(s) are generally placed adjacent an end of the capillary channel, which may be an end where fluid ultimately exits the capillary channel. The placement of the structural members is such as to achieve the intermittent movement of fluid between the linear array and the mixing area without fluid exiting the capillary channel. The structural member(s) are placed within about 1 to about 1000 microns, within about 10 to about 500 microns, within about 20 to about 200 microns, within about 50 to about 100 microns, of the end of the capillary channel.

The materials from which the structural members may be fabricated may be naturally occurring or synthetic or modified naturally occurring. The material should be compatible with the fluids that are in contact with the interior of the capillary channel. Thus, the material should not be reactive with or in any way cause deterioration of such fluids. The material may be homogeneous or heterogeneous, that is, the material may comprise a single component or it may comprise multiple components in the form of layers, composites, laminates, blends, photo-defined polymers, and the like. The material may be the same as or different from the material from which the housing is fabricated.

In one approach, the structural member and the capillary housing are formed from the same material. In one approach, the capillary housing and the structural member are manufactured from the same material as an integral system as discussed in more detail below.

Examples of structural members that may be employed in the present apparatus and methods include, by way of illustration and not limitation, those structures described in U.S. Pat. No. 4,426,451 (Columbus), U.S. Pat. No. 5,912,134 (Shartle 1), U.S. Pat. No. 6,488,827 (Shartle 2), U.S. Pat. No. 5,230,866 (Shartle, et al., 1), U.S. Pat. No. 6,521,182 (Shartle, et al., 2), U.S. Pat. No. 6,103,199 (Bjornson, et al.), U.S. Pat. No. 5,693,233 (Schembri), and U.S. Pat. No. 5,104,813 (Besemer, et al.), the relevant disclosures thereof are incorporated herein by reference.

In general, the material for the housing should provide physical support for the chemical compounds that are deposited on an interior surface of the housing or synthesized on an interior surface of the housing in situ from subunits. The materials should be of such a composition that they endure the conditions of a deposition process and/or an in situ synthesis and of any subsequent treatment or handling or processing that may be encountered in the use of a particular array.

Typically, the housing material is transparent or comprises a viewing area that is transparent. By "transparent" is meant that the substrate material permits signal from features on an interior surface of the substrate to pass therethrough without substantial attenuation and also permits any interrogating radiation to pass therethrough without substantial attenuation. By "without substantial attenuation" may include, for example, without a loss of more than about 40% or more typically without a loss of more than about 30%, about 20% or about 10%, of signal. The interrogating radiation and signal may for example be visible, ultraviolet or infrared light. In certain embodiments, such as for example where production of binding pair arrays for use in research and related applications is desired, the materials from which the substrate may be fabricated should ideally exhibit a low level of non-specific binding during chemical reactions such as, e.g., hybridization events. Alternatively, the material may be opaque if the covering forming the top of channel comprising the linear array is removed or opened prior to a scanning for optical signal or if non-optical detection methods are employed such as radiation.

Particular plastics finding use for the housing include, for example, flexible or rigid forms of polyethylene, polypropylene, polytetrafluoroethylene (PTFE), e.g., TEFLON.RTM., polymethylmethacrylate, polycarbonate, polyethylene terephthalate, polystyrene or styrene copolymers, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneamines, polyarylene sulfides, polysiloxanes, polydimethylsiloxanes, polyimides, polyacetates, poly etheretherketone (PEEK), and the like, either used alone or in conjunction with another material or materials. The housing may be rigid or flexible.

Suitable rigid materials may include glass, which term is used to include silica, and include, for example, glass such as glass available as Bioglass, and suitable rigid plastics and resins, and so forth. Rigid plastics include, for example, polymers such as, e.g., poly (vinyl chloride), polyacrylamide, polyacrylate, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinyl butyrate), etc., either used by themselves or in conjunction with other materials.

In one exemplary embodiment the structural member is in the form of a photo-defined polymer such as, for example, a polyamide and the like. Other photopolymers suitable for the present invention will be familiar to those skilled in the art in view of the teaching herein.

The structural member may be integral with the interior of the housing of the present device or it may be separate therefrom. The structural member may comprise a single element or multiple elements that cooperate to narrow the passage of the capillary channel. The structural member may take any of a number of geometrical forms depending on the above considerations. Such forms include, for example, cylindrical, rectangular, triangular, and the like.

In one embodiment, the structural member is a pair of rectangular photolithographically defined barriers. These barriers form capillary channels smaller in dimension that the capillary channel housing the linear array and, therefore, act as a capillary leash to hold onto the fluid as centrifugal force is applied to the linear array. Alternatively, a neck in the capillary channel may be formed; the neck is integral with the interior of the capillary channel. The neck serves the same function as the aforementioned pair of barriers.

Microfluidic systems are often fabricated using photolithography, wet chemical etching, and other techniques similar to those employed in the semiconductor industry. The resulting devices can be used to perform a variety of sophisticated chemical and biological analytical techniques.

In one specific embodiment of the present apparatus, the channel is a conduit by which a sample may contact a linear array comprising a plurality of features for conducting chemical reactions. The channels, and thus the linear array, may be straight, curved, serpentine, labyrinth-like or other convenient configuration comprised of separate tubes or part of a monolithic, often planar, substrate. The cross-sectional shape of the channel is not critical and may be circular, ellipsoid, square, rectangular, triangular and the like. The inside of the channel may be coated with a material for strength, for enhancing or reducing electrokinetic flow, for enhancing detection limits and sensitivity, and so forth. Exemplary of coatings are silylation, polyacrylamide (vinyl bound), methylcellulose, polyether, polyvinylpyrrolidone, and polyethylene glycol, polypropylene, Teflon.TM. (DuPont), Nafion.TM. (DuPont), and the like may also be used.

The channel may comprise at least one entry port, namely, any site at which a liquid may be introduced into a device having one or more channels. The entry port may be a well or simply the terminus of a channel that opens any place on the device such as at an edge. The channel may comprise at least one port from which fluid exiting the channel may travel to a collection chamber and the like.

The channel or the housing may also comprise a mixing area to which the fluid may move, without exiting the channel, in response to application of centrifugal force to said housing such as by rotation of the housing comprising the channel. As intermittent centrifugal force is applied to the housing, the fluid moves back and forth between the mixing area and the capillary channel. The mixing area may be a portion of the capillary channel or it may be independent thereof but in fluid communication with the capillary channel. The dimensions of the mixing area are dependent on the dimensions of the capillary channel, the dimensions of the structure, the amount of fluid, and so forth. The dimensions of the mixing area for capillary channels are about 10 to about 10,000 microns, about 50 to about 5,000 microns, about 100 to about 2,000 microns, about 500 to about 1,000 microns. The placement of the mixing area is such as to achieve the intermittent movement of fluid between the linear array and the mixing area without fluid exiting the capillary channel. The mixing area is placed within about 10 to about 10,000 microns, about 50 to about 5,000 microns, about 100 to about 2,000 microns, about 500 to about 1,000 microns, of the end of the capillary channel.

Microfluidic devices generally include one or more channels fabricated on or within the devices, for example, within the devices. The devices also can include reservoirs, fluidly connected to the channels, which can be used to introduce materials into the channels to contact a linear array contained in the channel. Microfluidic systems have a number of advantages over conventional chemical or physical laboratory techniques. For example, microfluidic systems are particularly well adapted for analyzing small sample sizes, typically making use of samples on the order of nanoliters and even picoliters. The substrates may be produced at relatively low cost, and the channels can be arranged to perform numerous specific analytical operations, including mixing, dispensing, valving, reactions, detections, electrophoresis, and the like. The analytical capabilities of such microfluidic systems may be enhanced by increasing the number and complexity of network channels, reaction chambers, and the like. However, in its simplest form for the purposes of the present invention involving linear arrays, the microfluidic system is often less complex.

The length of the linear array as manufactured may be a fixed length determined by the number of features of the linear array. The number of features is related to the nature of the features, the nature of the analytes, the complexity of the biological or clinical questions being investigated, the number of quality control features desired, and so forth. A typical linear array may contain more than about ten, more than about one hundred, more than about one thousand, more than about ten thousand, more than about twenty thousand, etc., more than about one hundred thousand, features and so forth.

The housing comprising the linear array may be prepared in a number of ways. The following discussion is by way of illustration and not limitation. In one approach, the linear array is synthesized or deposited on the surface of a housing substrate and the area comprising at least the linear array is enclosed to form a channel comprising the linear array and also a mixing area or chamber if desired. Enclosure may be attained using an appropriate material to cover the channel and then sealing to form the housing. The apparatus may be fabricated using other convenient means, including conventional molding and casting techniques, extrusion sheet forming, calendaring, thermoforming, and the like. For example, with apparatus prepared from a plastic material, a silica mold master, which is negative for the network structure in the planar substrate of one plate can be prepared by etching or laser micromachining. In addition to having a raised ridge, which forms the channel in the substrate, the silica mold may have a raised area that provides for one or more cavity structures in the planar substrate. Next, a polymer precursor formulation can be thermally cured or photopolymerized between the silica master and support planar plate, such as a glass plate.

In one embodiment, the linear array may be synthesized or deposited on the surface of a flexible material or substrate in the dimensions desired. For example, for a microarray the chemical compounds comprising the linear array are synthesized or deposited in an area that corresponds to capillary dimensions. The flexible substrate may be substantially flat along the area of synthesis or deposition or there may be a groove, depression, or the like in the housing substrate where the linear array is placed. This area of deposition or synthesis is ultimately enclosed to form a channel having the linear array therein. See, for example, U.S. patent application Ser. No. 10/037,757, entitled "Chemical Arrays" by Schembri, et al., filed Oct. 18, 2001, published as U.S. Patent Publication No. 20030108726 and U.S. patent application Ser. No. 10/032,608, entitled "Chemical Arrays", by Lefkowitz, et al., filed Oct. 18, 2001, published as U.S. Patent Publication No. 20030077380, the relevant disclosures of which are incorporated herein by reference.

Enclosing the housing to form the channel comprising the linear array and the mixing area or chamber, if one is included, may be accomplished in a number of ways. One important consideration in forming the linear array housing in general, and enclosing the housing in particular, is to avoid damage to the linear array on the surface of the housing substrate. In one approach, for example, the substrate is a flexible material that is folded over to enclose the housing to form the channel. After folding, the flexible material is sealed to itself in an area outside the area of the channel and mixing area. Sealing may be achieved by application of heat, adhesives, and so forth.

In an alternate approach, a separate material may be placed over the substrate comprising the linear array and the mixing area. The separate material is sealed to the substrate to enclose the housing to form the channel with the linear array therein and the mixing area. The separate material may be sealed to the substrate as discussed above. The separate material may have the same composition as the substrate or a composition that is different from the substrate. The separate material may be flexible or rigid.

The flexible substrate may be a plastic, that is, any synthetic organic polymer of high molecular weight (for example at least 1,000 grams/mole, or even at least 10,000 or 100,000 grams/mole. In one embodiment in accordance with the above disclosure, the flexible substrate may have a number of different layers. A base layer forms the greatest thickness and may consist of any flexible plastic such as a polyolefin film (such as polypropylene, polyethylene, polymethylpentene) or polyetheretherketone, polyimide, any of the fluorocarbon polymers or other suitable flexible thermoplastic polymer film. The material of the base layer is best selected to provide stable dimensional, mechanical, and chemical properties as well as severability. The flexible substrate may also include an optional reflective layer and a transparent layer. The reflective layer may be aluminum, silver, gold, platinum, chrome or other suitable metal film deposited by vacuum deposition, plasma enhanced chemical vapor deposition or other means onto the base layer or an optional intermediate bonding layer. Alternatively, the reflective layer may be constructed using multiple dielectric layers designed as a dielectric Bragg reflector or the like. A bonding layer, if used, may be any suitable material that is flexible at the thickness used and bonds to the base layer and/or the reflective layer. The bonding layer may have a thickness of less than about 50 nm, or even less than about 20, about 10, about 5 or about 1 nm and typically more than about 0.1 or about 0.5 nm). A glass layer (which term is used to include silica) may be deposited onto the reflective layer by sputtering, plasma enhanced chemical vapor deposition or similar techniques such as described in. A glass layer may optionally be used without a reflective layer. In the above configuration of the flexible substrate, the use of a glass layer allows the use of conventional chemistries, as discussed above, for substrate coating, feature fabrication, and array usage (for example, hybridization in the case of polynucleotide arrays). Such chemistries are well known for arrays on glass substrates, as described in the references cited herein and elsewhere. Furthermore, using a reflective layer not only can provide the useful characteristics mentioned in the above referenced patent application Ser. No. 09/493,958, but can avoid undesirable optical characteristics of the plastic base layer (for example, undesirable fluorescence, and in some instances, excessive heating and possible melting of the substrate). This allows for the ability to use base layers of a material that may have a high fluorescence and/or high absorbance of incident light. Use of a non-reflective opaque layer (for example, a suitably dyed plastic or other layer) in place of reflective layer also allows the use of the foregoing materials for a base layer although in such a case some heat may then be generated in the opaque layer.

The mechanism for causing a pressure differential on an intermittent basis to move fluid back and forth within the capillary channel and a mixing area may be any suitable mechanism. In one embodiment the mechanism is one that generates centrifugal force and may be any mechanism that can intermittently generate centrifugal force on the interior of the housing. In one embodiment the mechanism comprises a rotatable support upon which one or more of the housings are mounted. The rotatable support may be any structure that provides support for the linear arrays and that is capable of rotation about an axis, which may be a central axis. The rotatable support may be, for example, a circular tray such as a carousel or the like. The circular tray and mechanism may be similar in design to that employed, for example, in the Agilent G2505 Scanner, Agilent Technologies Inc., Palo Alto Calif.

The rotatable support may have a surface for receiving and holding the housings for the linear arrays. Thus, the surface of the rotatable support generally has a plurality of retaining elements for retaining the linear array housings on the surface. The design of the retaining elements is dependent on the nature of the housing for the linear array. The retaining elements should retain the housings sufficiently so that, during rotation of the support and other manipulations, the linear arrays remain securely on the surface. Such retaining elements include, for example, grooves in the surface of the rotatable support, elevated slots on the surface of the support, and so forth. The retaining elements may be disposed around the axis of rotation of the rotatable support. The orientation of the retaining elements should be such that the housings for the linear arrays are situated so that operation of the present apparatus to mix fluids in the channel comprising the linear array and to remove fluids from the channel as desired may be accomplished by rotation of the rotatable housing. In some circumstances, the orientation of the retaining elements, and thus the linear arrays, should also allow accurate analysis of the reaction results.

The dimensions of the rotatable support depend on the nature of the housings of the linear arrays, the required distance of the housings from the axis of rotation of the support, and so forth. The dimensions of the rotatable support should be governed by practical considerations such as the overall size of the device, the size of the motor required to rotate the support and the like. The dimensions of the rotatable support should be large enough to accommodate the number of linear arrays on its surface, but not so large as to be impractical. For linear microarrays of approximately 20 mm in length, the dimensions of the rotatable support that is circular may be about 25 mm to about by 35 mm, about 28 to 30 mm.

The housings for the linear arrays should be disposed from the axis of rotation of the rotatable support so that the linear array exit port is a sufficient distance from the axis of rotation so that fluid in the linear array may be moved through and out of the linear array. The housings for the linear array are about 5 to about 100 microns, about 10 to about 50 microns, from the axis of rotation of the rotatable support.

The number of retaining elements, and thus the maximum number of linear array housings, on the surface of the rotatable support is about 1 to about 1,000, about 5 to about 500, about 10 to about 100, about 20 to about 80.

The rotatable support is driven by a suitable driving mechanism such as a motor and the like, which is capable of rotating the rotatable support in accordance with the present invention. Accordingly, the driving mechanism is adapted to rotate the rotatable support at speeds required to mix fluids in the channel comprising the linear array and the to subsequently overcome capillary forces in the microchannels of the linear arrays. In some embodiments, it may be desirable to rotate the rotatable support in a step-wise fashion to index the linear arrays to, for example, an examining device. A motor may be, for example, a stepping-type motor, a servo-type motor, and so forth. The driving mechanism may be in communication with a system controller, which provides control over the speed of rotation of the rotatable support, indexed movement of the rotatable support, and so forth. The speed of rotation of the rotatable support is dependent on a number of factors including, for example, the nature of the function being performed at any point in time, and so forth. For removal of fluid from the interior of the linear array, the rotatable support is rotated at a speed great enough to produce a centrifugal force that overcomes the capillary forces on the fluid. This speed is dependent on the surface tension, etc., of the fluid, the capillary dimensions of the channel, the capillary dimensions of the structure, the distance of the capillary from the center of rotation, and the like. The pressures for achieving the desired results are dependent on the mechanical properties of the fluids, the mechanical properties of the surface of the capillary channel, the dimensions of the capillary, and so forth. The pressure for mixing varies from about 1 to about 100 inches of water, from about 2 to about 50 inches of water, from about 4 to about 30 inches of water. Where the pressure generated is centrifugal force, the speed of rotation of the linear array may be employed to achieve the desired effect. For mixing, the speed of rotation may be intermittently varied.

The driving mechanism may provide for indexing of the linear arrays on the surface of the rotatable support. To assist in the indexing function, the linear array housings may comprise an identification code. A suitable reading device is employed for reading the identification code. The reading device is incorporated into the present apparatus or is separate from the present apparatus. The reading device is located so as to provide an accurate reading of the identification code of the linear array housings. The code is read at a time best suited for providing accurate identification. Thus, the reading device may read the code when each of the housings for the linear arrays is in place on the rotatable support. The code may also be read while the housings are loaded onto the rotatable support. Information read from the identification code is fed to a system controller for the apparatus and correlated with the indexed position of the housing on the rotatable support. In this way the identity and location of each of the linear array housings can be tracked and the processing of each linear array may be linked to the identification code. Such information may be loaded into a data storage database for use by other systems.

An example of a specific embodiment of an apparatus in accordance with the present invention is discussed next with reference to the attached drawings. Referring to FIGS. 1 and 2, apparatus 10 is depicted and comprises housing 12 with capillary channel 14 and structural members 16 adjacent an end of channel 14. Within channel 14 are a linear array of features 18. Entry port 22 is approximate one end of housing 12 and mixing area or chamber 24 is approximate an opposing end of housing 12.

Another example of a specific embodiment of an apparatus in accordance with the present invention is set forth in FIG. 3. Apparatus 30 is depicted and comprises housing 32 with capillary channel 34 having narrowing section 36 (or neck) as a structural member adjacent an end of channel 34. Within channel 34 are a linear array of features 38 separated by regions 40 that are free of features.

Another example of a specific embodiment of an apparatus in accordance with the present invention is set forth in FIG. 4. Apparatus 50 is depicted and comprises housing 52 with capillary channel 54 having structural members 56 (that form a narrowing section or neck) adjacent an end of channel 54. Within channel 54 are a linear array of features 58 separated by regions 60 that are free of features.

Referring to FIGS. 1 and 2, features 18 of the linear array are non-diffusively bound an interior surface of channel 14. Inter-feature regions 20 separate features 18. A typical linear array may contain from about 100 to about 100,000 features. At least some, or all, of the features are of different compositions (for example, when any repeats of each feature composition are excluded the remaining features may account for at least about 5%, about 10%, or about 20% of the total number of features). Each feature carries a predetermined moiety (such as a particular polynucleotide sequence), or a predetermined mixture of moieties (such as a mixture of particular polynucleotides).

A linear array of features is a one-dimensional array of features bound in a non-diffusive manner to a surface. By the term "non-diffusive" is meant that the molecules that make up the individual fe