Abstract:
A method and apparatus for analyzing nucleic acids includes immobilizing nucleic probes at specific sites within a microchannel structure and moving target nucleic acids into proximity to the probes in order to allow hybridization and fluorescence detection of specific target sequences.

Description:
This is a continuation of application Ser. No. 08/848,553, filed Apr. 28, 1997, abandoned. 
    
    
     This invention was made with government support under Contract No. DE-AC05-840R21400 awarded by the U.S. Department of Energy to Lockheed Martin Energy Systems, Inc. and the government has certain rights in this invention. 
    
    
     BACKGROUND OF THE INVENTION 
     Field of The Invention 
     The present invention relates generally to medical and/or biological testing and devices for performing same, and more particularly, to a method and apparatus for analyzing minute amounts of nucleic acids for the presence of specific nucleotide sequences. Single-strand DNA probes are bound to specific regions of microchannels in a glass microchip device. Sub-microliter volumes of nucleic acid solutions, buffers and other reagents are transported through the channels under electrokinetic or hydraulic control. Hybridization of target nucleic acid sequences to complementary probes is detected using either fluorescent labels or intercalating fluorescent dyes. 
     Description of the Related Art 
     Hybridization analysis is typically performed in microtiter plate wells or on planar surfaces that contain arrays of DNA probes. Chemical manipulations are required to bring about a hybridization test and to detect the results. These manipulations presently include washing or dipping planar arrays into the appropriate chemicals. 
     The aforementioned procedures suffer from many drawbacks. For example, they are wasteful of expensive reagents and limited sample volumes. Moreover, they are generally not compatible with efficient automation strategies and thus tend to be time consuming. 
     A continuing need exists for methods and apparatuses that limit the use of expensive reagents and priceless samples, while simplifying the overall procedures to require smaller samples and fewer processing steps. 
     SUMMARY OF THE INVENTION 
     An object of the present invention is to provide a method and apparatus for analyzing nucleic acids which simplifies chemical manipulations required to bring about a hybridization test when performing DNA diagnostics in biomedical, forensic, and research applications. 
     Another object of the present invention is to provide a method and apparatus for analyzing nucleic acids which minimizes the use of expensive reagents and limited sample volumes. 
     Another object of the present invention is to provide a method and apparatus for analyzing nucleic acids which avoids the necessity of pre-labeling a target DNA and increases the sensitivity of hybrid detection by reducing background fluorescence due to non-specific surface adsorption of labeled target DNA. 
     Still another object of the present invention is to provide a method and apparatus for analyzing nucleic acids which significantly extend the usefulness of hybridization diagnostics by allowing its application to much smaller samples and facilitating automated processing. 
     These and other objects are met by providing an apparatus for analyzing nucleic acids which includes a microchip having a microchannel structure formed therein, at least one portion of the microchannel structure having at least one site capable of affixing thereto a probe, and a plurality of reservoirs in communication with the microchannel structure for introducing at least one of, or a mixture of, a reagent, analyte solution, and buffer. 
     In another aspect of the invention, a method of analyzing nucleic acids includes bonding oligonucleotide probes to a microchannel formed in a microchip, adding target nucleic acids and fluorescent stains to the microchannel, and detecting hybridization by fluorescence staining of double-stranded DNA. 
     These together with other objects and advantages which will be subsequently apparent, reside in the details of construction and operation as more fully hereinafter described and claimed, with reference being had to the accompanying drawings forming a part hereof, wherein like numerals refer to like elements throughout. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a schematic view of an apparatus for analyzing nucleic acids according to a preferred embodiment of the present invention; 
     FIGS. 2 and 3 are schematic views of different arrangements of nucleic acid hybridization probes in microchannels; 
     FIG. 4 is a schematic view of a microchip and microchannel structure according to another preferred embodiment of the present invention; 
     FIG. 5 is a schematic view of a microchip of the present invention; 
     FIG. 6 is a photomicrograph showing discrimination of target and non-target DNA at the intersection of microchannels in the inset area of FIG. 5 after dsDNA staining with fluorescent dye; 
     FIG. 7 is a schematic view of another apparatus for analyzing nucleic acids according to a preferred embodiment of the present invention; and 
     FIG. 8 shows fluorescence image profiles of two probe channels after ds-DNA staining with fluorescent dye. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     Referring to FIG. 1, a microchip  16  includes a glass substrate  18  and a cover plate  20  which covers a microchannel structure  22  formed in the upper surface of the substrate  16 . The cover plate  20  is permanently bonded to the substrate  18 . Both the substrate  18  and cover plate  20  are preferably made of clear glass, and the substrate may preferably be made from a standard microscope slide. Alternative construction materials could include plastics (such as polypropylene, polycarbonate, or polymethylmethacrylate), silicon, or sapphire. 
     The microchannel structure  22  is formed using standard photolithographic techniques, and includes a longitudinal microchannel manifold portion  24 , a first transverse microchannel portion  26  forming an intersection  28  with the longitudinal portion  24 , and a second transverse microchannel portion  30  forming an intersection  32  with the longitudinal portion  24 . 
     First and second reservoirs  34  and  36  are in fluid communication with opposite ends of the longitudinal portion  24 . The opposite ends act as ports to introduce the contents of the reservoirs  34  and  36  into the microchannel structure  22 . Each reservoir can be a cylindrical container open at its opposite axial ends, with the ends of the longitudinal portion  24  being in fluid communication with the bottom of the container. 
     Third and fourth reservoirs  38  and  40  are in fluid communication with opposite ends of the first transverse portion  26 . The opposite ends act as ports to introduce the contents of the reservoirs  38  and  40  into the microchannel structure  22 . Each reservoir  38  and  40  is similar in construction to the other reservoirs, with the ends of the first transverse portion being in fluid communication with the bottom of each respective reservoir  38  and  40 . 
     Fifth and sixth reservoirs  42  and  44  are in fluid communication with opposite ends of the second transverse portion  30 . The opposite ends act as ports to introduce the contents of the reservoirs  42  and  44  into the microchannel structure  22 . Each reservoir  42  and  44  is similar in construction to the other reservoirs, with the ends of the second transverse portion being in fluid communication with the bottom of each respective reservoir  42  and  44 . 
     One or more types of single-stranded DNA probes  46  are attached at individual sites within the microchannel portion  24  of the microchannel structure  22 . The design and fabrication of microchips and the electrokinetic transport of fluids through the microchannels is described in U.S. Ser. No. 08/283,769, filed Aug. 1, 1994, U.S. Pat. No. 6,001,229 hereby incorporated by reference. The microchips described therein include planar, glass substrates into which the microchannels are etched photolithographically. The reservoirs typically hold analyte solutions, buffers, reagents, etc. Typical microchannel dimensions are 10 μm by 50 μm (depth×width), although channel widths of 1 μm to &gt;100 μm and channel depths of &lt;1 μm to &gt;100 μm may be used. Voltages are applied to solutions as described in the aforementioned application to produce electroosmotic flow of fluids or electrophoretic migration of charged species through the channels. Alternatively, pressure (or vacuum) may be applied to one or more fluid reservoirs to cause reagent flow through the channels. 
     The individual DNA probes may be arranged in a linear pattern, as shown in FIG.  2 . An alternative embodiment is shown in FIG. 3, wherein the  46 ′ are arranged in a two-dimensional array in a widened area  48  of the channel portion  24 ′. Fluid flow is in the direction indicated by arrows. 
     Typically, oligonucleotide probes ten to thirty nucleotides long are used for hybridization analysis, although much longer probes, such as DNA restriction fragments or cDNA sequences of &gt;100 nucleotide length, may be used in certain applications. 
     Oligonucleotide probes may be immobilized by covalent chemical linkage to the surface. In general, such linkage involves derivatization of the glass surface with a silane coupling agent, such as 3-aminopropyltriethoxysilane or 3-glycidoxypropyltrimethoxysilane. An oligonucleotide probe bearing an alkylamine group at the 5′ or 3′ end may then be linked to the surface by direct reaction of its terminal amine with a silane epoxy group or by cross linking the silane and oligonucleotide amines using glutaraldehyde or other amine-reactive bi-functional compounds. 
     Other immobilization method may also be used. For example, surface-immobilized avidin or streptavidin may be used to bind biotinylated probes. Non-covalently adsorbed oligonucleotides on glass surfaces have also been shown to hybridize to target sequences. 
     In the preferred fabrication method, the probes are attached to the open microchip channels and the cover plate is then bound to the substrate by a low temperature technique which does not damage the biomolecules. Such a low temperature bonding technique is described in application Ser. No. 08/645,497, abandoned, entitled “Low Temperature Material Bonding Technique” by J. M. Ramsey, R. S. Foote, and H. Wang, which is incorporated herein by reference. Individual probes may be applied to specific sites in the channels by micro-pipeting or other means, such as ink-jet printing. The separation of individual probes may be facilitated by preparing the surface with a pattern of reactive, hydrophilic sites separated by non-reactive, hydrophobic areas. For example, the glass surface may be treated with an alkyltrialkoxysilane to produce a non-reactive, hydrophobic surface. Photolithography and chemical etching or laser ablation may be used to remove the silane layer and expose the glass substrate in a pattern of separated spots. These spots may then be treated with a silane coupling agent as described above to produce reactive, hydrophilic spots. An aqueous probe solution applied to an individual spot would be confined to its hydrophilic site and thus prevented from mixing with different probe solutions in adjacent spots. The intervening hydrophobic regions would also prevent probe mixing in the case of the other immobilization methods described above. 
     Alternatively, the probes may be attached to specific sites in the channels after standard high-temperature cover plate bonding. Three methods of achieving this are provided as examples: 
     (1) The functional group of the silane linker (e.g., the amino function of 3-aminopropylsilane)may be blocked with a photolabile protective group. The silane linkers are then de-protected at specific positions in the channel by exposure to light through the cover plate using a photolithographic mask or focused beam. Cross linkers and probes passed through the channel would react only at de-protected sites. A series of separate de-protection and addition steps are used to attach a number of different probes to individual sites. 
     (2) An array of oligonucleotide probes may be photochemically synthesized in situ in a parallel fashion. 
     (3) A channel manifold may be designed to allow the addition of an individual probe to a given branch or segment of the manifold by controlling fluid flows. 
     In the preferred methodology, nucleic acids, buffers and dyes are electrokinetically driven through the microchannels containing the immobilized probes. For example, the following sequence of operations can be used with the device schematically illustrated in FIG.  4 . As seen in FIG. 4, a microchip  50  includes a microchannel structure  52  connected to a nucleic acid sample reservoir  54 , a buffer reservoir  56 , a dye reservoir  58 , dye buffer reservoir  60 , and waste reservoir  62 . A hybridization chamber  64  is disposed in the microchannel structure  52  between first and second transverse portions  66 ,  68  of the microchannel structure. 
     A voltage is applied between reservoir  54  which contains the nucleic acid sample being analyzed and reservoir  56  containing nucleic acid buffer. For buffers containing a high NaCl concentration (desirable for rapid nucleic hybridization) the polarity of reservoir  56  is positive relative to reservoir  54  and the negatively charged nucleic acids electrophoretically migrate from reservoir  54  to reservoir  56 , passing through the hybridization chamber  64 . Alternatively, a nucleic acid solution containing a low salt concentration may be electroosmotically transported into the hybridization chamber by applying a positive voltage at reservoir  54  relative to reservoir  56 . Because electroosmotic flow toward reservoir  56  is high relative to electrophoretic migration toward the positive electrode, the net movement of nucleic acids will be toward reservoir  56  in the later case. The use of electroosmotic flow versus electrophoretic migration will depend on a number of factors, and may vary depending on the type of sample being analyzed. The term “electrokinetic transport” includes both electroosmotic flow and electrophoretic migration. 
     After the DNA sample reaches equilibrium over the probe sites, the voltage may be discontinued while hybridization occurs. A double-strand-DNA-specific (dsDNA-specific) fluorescent dye is then electrokinetically transported through the hybridization chamber  64  by applying voltages to fluid reservoir  58  which contains a dye and reservoir  60  containing a dye buffer. Because high salt concentrations are not normally required or desirable for this step, electroosmotic flow is the preferred method of dye addition and the polarity of reservoir  58  will normally be positive relative to reservoir  60 . Several fluorescent double-strand-specific nucleic acid stains are commercially available. Many of these stains are positively charged so that their electrophoretic migration will be in the same direction as the electroosmotic flow. 
     Alternatively, the nucleic acids being analyzed may be pre-labeled with fluorescent groups by well known procedures. Although this later method can lead to higher background fluorescence, it may be preferred in cases where probes contain self-complementary sequences that can result in stable duplex formation and dye binding by the probe itself. 
     Variations in the chip design and analysis procedure are possible. For example, electrokinetically driven washing steps may be included before and/or after the dye addition step by applying appropriate voltages between the buffer reservoirs and a waste reservoir  62 . Nucleic acid and dye solutions might also be added simultaneously to the hybridization chamber. As an alternative to electrokinetically driven fluid manipulation, hydraulic pressure or vacuum may be applied to appropriate reservoirs to control the flow of solutions through the microchannels. 
     After completion of the hybridization and dsDNA staining steps, if used, the hybridization chamber is examined for the presence of fluorescently labeled sites by illumination with exciting light through the cover plate. An epifluorescence microscope and CCD camera may be used, as described below, to obtain a fluorescence image of the entire chamber or portion thereof. Scanning confocal fluorescence microscopy may also be used. 
     The following examples incorporate the apparatus and methodology of the present invention. Each involves the steps of (1) covalently bonding oligonucleotide probes to microchannels, (2) adding target nucleic acids and fluorescent stains to microchannels by electrokinetic flow, (3) detecting hybridization by fluorescence staining of double-stranded DNA, and (4) discriminating target and non-target nucleic acids. 
     EXAMPLE 1 
     A 16-mer oligodeoxynucleotide probe sequence containing a 5′-(6-aminohexyl)phosphate [H 2 N—CH 2 ) 6 -5′-pCGGCACCGAGTTTAGC-3′] (SEQ ID NO: 1) was covalently attached to the hybridization chamber of a prototype microchip similar to that shown in FIG. 4 by glutaraldehyde cross linking with the 3-aminopropylsilane-derivatized glass surface. A complementary 16-mer (target sequence) oligodeoxynucleotide in 6×SSC buffer was then electrophoretically added to the hybridization chamber by applying 0.5 kV between reservoir  56  and reservoir  54  (positive electrode at reservoir  54 ) for thirty minutes. A dsDNA-specific fluorescent dye (TOTO-1, Molecular Probes) in 10 mM Tris-borate buffer, pH 9.2, was then electroosmotically added to the chamber by applying 1.0 kV between reservoir  60  and reservoir  58  for 30 minutes. The chip was examined by video microscopy using laser excitation (514 nm) of fluorescence. Bright fluorescence due to the dsDNA-bound dye was observed in the hybridization chamber relative to channels not exposed to the target DNA. The image was recorded on video tape. 
     In a subsequent similar experiment using the ds-DNA specific dye, PicoGreen (Molecular Probes), quantification by CCD imaging and analysis showed a 10-fold increase in fluorescence intensity when staining was carried out after hybridization of the target DNA, relative to the intensity observed by staining prior to the hybridization step. 
     EXAMPLE 2 
     The 16-mer oligonucleotide probe of Example 1 was uniformly bound to the channels of a cross-channel chip shown schematically in FIG. 5 by glutaraldehyde cross-linking. Solutions (50 μM) of the complementary (target sequence) 16-mer oligodeoxynucleotide (T) and a non-complementary (non-target sequence) 16-mer oligodeoxynucleotide (N) in phosphate-buffered saline (PBS) were then added to separate channels as indicated in FIG. 5, by applying suction at W for 10 minutes. The channels were then washed with buffer and dsDNA-specific dye solution (PicoGreen, Molecular Probes) was added to all channels for five minutes. The cross-channel intersection was examined by epifluorescence microscopy using a mercury lamp illumination source and FITC filters. A 1.0 second CCD exposure, shown in FIG. 6 as the insert of the broken line area of FIG. 5, showed intense fluorescence (dark regions) in the channel exposed to target DNA relative to that of channels exposed to non-target DNA or buffer. 
     In a similar experiment using laser induced fluorescence imaging, as described in application Ser. No. 08/800,241, U.S. Pat. No. 6,056,859, entitled “Method and Apparatus for Staining Immobilized Nucleic Acids” by J. M. Ramsey, R. S. Foote and S.C. Jacobson, incorporated herein by reference, signal intensity from channels exposed to target DNA was 10-fold greater than from channels exposed to non-target DNA or buffer. 
     EXAMPLE 3 
     Two 16-mer probes [H 2 N—(CH 2 ) 6 -5′-GCTAAACTCGGTGCCG-3′ (Probe 1)] (SEQ ID NO: 2) and [H 2 N—(CH 2 ) 6 -5′-pCGGCACCGAGTTTAGC-3′ (Probe 2)] (SEQ ID NO: 1) were immobilized in separate channels of a cross-channel chip as indicated in FIG.  7 . In FIG. 7, the “T” reservoir is for target DNA, “B” is for PBS buffer and “W” is for waste. 
     A solution of 16-mer oligonucleotide (50 nM oligonucleotide in PBS) complementary to Probe 1 was induced to flow through both channels for a total of 15 minutes by applying a vacuum at W. The channels were then washed with buffer and treated with a ds-DNA specific dye solution (PicoGreen, Molecular Probes) for two minutes. After washing with 10 mM Tris-HCL (pH 8), one mM EDTA (TE) buffer for one minute, the channels were examined for laser-induced fluorescence using an argon ion laser at 488 nm and 100 milliwatts power. Quantitation by CCD imaging, shown in FIG. 8, shows a 4 to 5-fold greater fluorescence in the Probe 1 channel than in the Probe 2 channel after subtraction of the background signal. 
     According to the above methods and apparatuses, hybridization analysis can be performed in a microchip structure that requires low instrumentation space and extremely low sample/reagent volumes. The electrokinetic transport of samples and reagents facilitates automation of sample/reagent manipulations. Moreover, the detection of hybridization using double-strand DNA-specific fluorescent dyes eliminates the target DNA labeling step associated with prior art techniques and increases detection sensitivity. 
     While the examples referred to above describe nucleic acid probes, the methodology and apparatuses could also be used for other uses including, but not limited to, immobilized antibodies for micro-immunoassays. Numerous biomedical applications can be envisioned. 
     While the various embodiments have referred to specific reservoirs containing specific reagents, buffers or samples, mixtures of two or more substances can be contained in individual reservoirs. For example, a reservoir can contain a mixture of reagent and buffer, buffer and sample, etc. 
     The many features and advantages of the invention are apparent from the detailed specification, and thus, it is intended by the appended claims to cover all such features and advantages of the invention which fall within the true spirit and scope of the invention. Further, since numerous modifications and variations will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation illustrated and described and accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the invention. 
     
       
         
           
             2 
           
           
             1 
             16 
             DNA 
             Artificial Sequence 
             
               Sequence source/note=“synthetic
      oligonucleotide construct containing a 6-amino hexyl phosphate
      modification at the 5′ end” 
             
           
            1
cggcaccgag tttagc                                                     16
 
           
             2 
             16 
             DNA 
             Artificial Sequence 
             
               Sequence source/note=“synthetic
      oligonucleotide construct containing a 6-amino-hexyl phosphate
      modification at the 5′ end” 
             
           
            2
gctaaactcg gtgccg                                                     16