Abstract:
A low-cost, easy to operate, three-phase tilting agitator for microarrays, including large area microarrays, provides experimentally verified improvements in hybridization intensity and uniformity. Motion is coupled from a single motor to a sample holder via three suspension tethers. The microarrays may be immersed in a water bath during agitation to maintain a temperature for the hybridization reaction. The use of traditional cover slips for the microarrays minimizes the volume requirement for target sample solution.

Description:
RELATED APPLICATION  
       [0001]     The present invention is related to Chinese patent (utility model) application No. 200420001127.7, filed on Apr. 19, 2004, the content of which is incorporated by reference herein in its entirety.  
       TECHNICAL FIELD  
       [0002]     The present invention relates to a reaction apparatus for microarrays, that is particularly suitable to large area microarrays, such as genome-wide DNA microarrays.  
       BACKGROUND ART  
       [0003]     DNA microarrays are two-dimensional arrays of reference DNA on glass membranes, microscope slides, or similar substrates. Microarrays are fabricated by spotting small volumes of solution containing reference (probe) DNA onto the substrate. In gene expression profiling assays, cDNA molecules originating from test and control samples competitively bind to the spotted probe molecules on a DNA microarray. The test and the control samples are labeled with two different fluorescent dyes to determine the intensity ratio with a fluorescence scanner. A ratio of one indicates the same expression level and a ratio different from one represents an up- or down-regulation of a respective gene. DNA microarrays can have surfaces covered by thousands of spots, and each spot can contain billions of cDNA probes corresponding to a particular known gene. The targets are poured onto the probe array, the targets hybridize with the complementary probes (if present in the array), and the array is washed to removed target that did not hybridize. This approach allows a parallel, semi-quantitative analysis of thousands of transcription levels in a single experiment. Although the discussion herein uses DNA microarrays as an example, microarrays may also be used for other types of affinity assays than DNA, for example, immunological assays, that rely on the hybridization of biological molecules.  
         [0004]     Microarray substrates are often conventional microscope slides with dimensions of 75 by 25 mm. Up to several thousand spots of oligonucleotides or cDNA proves with known identity cover the slide in a two dimensional grid. In a standard experimental set up, a buffered solution containing potential targets is sandwiched between a DNA microarray and a cover slip to form a reaction chamber with an area of several square centimeters and a height of only twenty to a hundred microns. The microarray assembly can be sealed in a humid chamber or placed in a water bath to prevent drying and/or control reaction temperature, and allowed to hybridize for a period of several hours. In such a configuration, diffusion is the only mechanism for DNA strands, or other targets, to move within the reaction chamber. However, diffusion is a notoriously slow process for molecules the size of DNA strands which may need to travel a distance of several centimeters to reach a microarray spot with a complementary probe. In such a case, the immediate vicinity of a probe spot can be quickly depleted, especially in the case of cDNA molecules representing genes with low expression.  
         [0005]     This diffusion limitation can lead to low signal-to-noise ratios when a microarray is read because only a fraction of the molecules present in the sample may get a chance to bind to their complimentary spots. Generally speaking, when a microarray&#39;s area reaches approximately 22 cm by 22 cm, it can be defined as a large area microarray. For large area microarrays, such as genome-wide DNA microarrays, the diffusion limitation and low signal-to-noise ratios are further exacerbated because of the longer travel distances for the target molecules.  
         [0006]     A solution to overcome the diffusion limitation and improve the reaction kinetics for better intensity and uniformity of hybridization is to agitate the target sample solution. The low height and large area of the reaction chamber formed by the microarray and the cover slip can make effective agitation difficult, especially for large area microarrays. Current approaches for agitation of the target sample solution include, for example: (i) microfluidic circulation, (ii) ultrasonic agitation, and (iii) contact with overlayed expanding and contracting air bladders. A drawback of microfluidic circulation is the requirement of three to five times as much target sample solution. The drawbacks of the ultrasonic and air bladder methods include cost and complexity of use, as well as the need for additional consumable materials. Advalytix AG of Brunnthal, Germany markets a line of products based on ultrasonic techniques. BioMicro Systems, Inc. of Salt Lake City, Utah, markets a line of products based on air bladder techniques. Both the ultrasonic and the air bladder techniques are difficult to scale up to handle large area microarrays.  
         [0007]     In view of the above discussion, it is very desirable to have a reaction apparatus for use with microarrays that is low cost, easy to use, and capable of effectively agitating large area microarrays.  
       SUMMARY OF THE INVENTION  
       [0008]     A low-cost, easy to operate, three-phase tilting agitator for microarrays, including large area microarrays, provides experimentally verified improvements in hybridization intensity and uniformity. Motion is coupled from a single motor to a sample holder via three suspension tethers. The microarrays may be immersed in a water bath during agitation to maintain a temperature for the hybridization reaction. The use of traditional cover slips for microarrays minimizes the volume requirement for target sample solution. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0009]      FIG. 1  illustrates a perspective view of an embodiment of the invention.  
         [0010]      FIG. 2  illustrates a top view of a suspension tether separation plate in an embodiment of the invention.  
         [0011]      FIG. 3   a  shows plots of suspension tether lengths above the tether separation plate in an embodiment of the invention.  
         [0012]      FIG. 3   b  shows plots sample plate attachment point heights according to an embodiment of the invention.  
         [0013]      FIGS. 4   a ,  4   b , and  4   c  illustrate a sample plate in three different extreme orientations.  
         [0014]      FIG. 5  illustrates a perspective view of another embodiment of the invention.  
         [0015]      FIG. 6  is a block diagram for a motor control system according to an embodiment of the invention.  
         [0016]      FIGS. 7   a  through  7   d  shows a hybridization result comparison between using microarray agitation according to an example of the present invention in a water bath and traditional microarray incubation without agitation in a water bath as an experimental control.  
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0017]     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, applications, published applications and other publications referred to herein are incorporated by reference in their entirety. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference.  
         [0018]     As used herein, “a” or “an” means “at least one” or “one or more.” 
         [0019]     Similar numerical references refer to similar features within the various drawings.  
         [0020]     Referring to  FIG. 1 , a sample holder  109  (in this embodiment, a sample plate  109 ) is suspended by three tethers  110   a ,  110   b , and  110   c  attached to sample plate  109  at attachment points  11   a ,  111   b , and  111   c , respectively. Although sample plate  109  is illustrated as a planar disc in this embodiment, the sample holder can be other structures such as trays, compartmented trays, single or multiple microarray cassette holders, or other types of container in other embodiments. Suspension tethers  110   a ,  110   b , and  110   c  pass through orifices  108   a ,  108   b , and  108   c , respectively, of the tether separation structure  106  (in this case a tether separation plate), where all three tethers are coupled to bearing  105 . Normally, but not necessarily, suspension tethers  110   a ,  110   b , and  110   c  are of substantially the same length. Normally, but not necessarily, orifices  108   a ,  108   b , and  108   c  are at substantially equal angular separations. Bearing  105  is coupled to a radial member  104  (in this embodiment, a radial arm), that is rotationally driven by motor  101  via shaft  103 . Motor  101  and tether separation plate  106  are coupled to structural support  113  via coupling members  102  and  107 , respectively. Structural support  113  is mounted on base  112 . In normal operation, a microarray  114  can be placed on sample plate  109 .  
         [0021]      FIG. 2  shows a top view of suspension tether separation plate  106 , with suspension tethers  110   a ,  110   b , and  110   c  coupled to bearing  105  as it rotates in a circular path. The lengths of suspension tethers  110   a ,  110   b , and  110   c  that extend above tether separation plate  106  are designated La, Lb, and Lc, respectively. An angle, theta  201 , measures the rotational position of bearing  105 , measured counterclockwise from the  108   a  orifice position.  FIG. 3   a  plots the sinusoidal variations of La, Lb, and Lc versus the angle, theta. Because each of the lengths of suspension tethers  110   a ,  110   b , and  110   c  are fixed, the larger the value of La, Lb, or Lc, the higher the heights of the attachment point  111   a ,  111   b , or  111   c , respectively, of sample plate  109 , relative to base  112 , as plotted in  FIG. 3   b .  FIGS. 4   a ,  4   b , and  4   c  show perspective views of the tilt of sample plate  109  when bearing  105  is positioned over orifices  108   a ,  108   b , and  108   c , respectively, of tether separation plate  106 . The rotation of bearing  105  coupled to radial arm  104  thus provides a three-phase, sinusoidal tilting of sample plate  109 , and microarray  114  resting on sample plate  109 . The three-phase, sinusoidal tilting effectively agitates the target solution of a microarray in a manner that increases toward the periphery of, and decreases toward the center of, sample plate  109 . In the embodiment illustrated, radial arm  104  is of such a length that bearing  105  passes substantially over orifices  108   a ,  108   b , and  108   c  as it rotates. In other embodiments radial arm  104  can be longer or shorter. In a further embodiment, bearing  105  has an adjustable radial position in order to control the amplitude of the tilting of sample plate  109 . In other embodiments, radial member  104  can be replaced with a disc to which bearing  105  can be coupled.  
         [0022]     Suspension tethers  110   a ,  110   b , and  110   c  can be made of any appropriate material, for example without exclusion: (i) single or multi-strand polymer, (ii) single or multi-strand natural fiber, (ii) single or multi-strand metal or metal alloy, (iv) single or multi-strand composite materials, or (v) chains made of polymer, metal, metal alloy, or composite materials. Suspension tethers  110   a ,  110   b , and  110   c  can be coupled to sample plate  109  at attachment points  111   a ,  111   b , and  111   c , respectively using any one of a variety of mechanical coupling techniques (including passing through a hole near the perimeter of sample plate  109 , and tying) that are well known to one of ordinary skill in the mechanical arts.  
         [0023]     In the preceding, exemplary embodiments, suspension tethers  110   a ,  110   b , and  110   c  are coupled to bearing  105  to prevent tangling as radial arm  104  rotates. In other embodiments, suspension tethers  110   a ,  110   b , and  110   c  can be coupled directly to a radial member.  
         [0024]     In some embodiments, orifices  108   a ,  108   b , and  108   c  of sample plate  106  are configured to reduce friction with and wear to suspension tethers  110   a ,  110   b , and  110   c . Such configurations can include, for example, contoured cross-sectional profiles, coating with a low friction material such as polytetrafluroethylene (PTFE), and/or the insertion of a low friction grommet. Although suspension tether separation structure  106  has been illustrated as a disc with three orifices,  108   a ,  108   b , and  108   c , in other embodiments equivalent structures for maintaining the separation of suspension cords  110   a ,  110   b , and  110   c  can be readily identified by one of ordinary skill in the art.  
         [0025]      FIG. 5  illustrates another embodiment, in which radial arm  104  of  FIG. 1  has been replaced by a disc  104  of  FIG. 5 , and there are three structural supports  113 . Sample plate  109  and suspension tethers  110   a ,  110   b , and  110   c  are water proof, so that microarray  114  may be immersed in a water bath to maintain a constant temperature during hybridization. The embodiment illustrated in  FIG. 5  can hold cassettes for one to twenty microarrays. The microarray area can range up to 22 cm by 22 cm, to enable genome-wide assays.  
         [0026]      FIG. 6  is a block diagram of a controller for controlling motor  604  in an embodiment where the motor is a stepper motor. An uninterruptible power supply  601 , having backup battery  606 , is used to maintain the agitation of a microarray in the event of a mains power failure. AC/DC power supply  602  converts mains AC power to the dc power required by motor driver  603 . Pulse adjuster  605  is used with motor driver  603  to control the speed of stepper motor  604 , as it is driven by motor driver  603 . Other embodiments can use other types of motors, for example without exclusion: (i) synchronous AC motors, (ii) brush-type DC motors; or (iii) brushless DC motors.  
         [0027]     Experimental comparisons of microarray hybridization reactions conducted with agitation by the present invention, and conducted with only diffusive target solution transport (i.e. no agitation) for control purposes, indicate substantial improvements in hybridization intensity and uniformity when conducted with the present invention.  
         [0028]      FIGS. 7   a  through  7   d  shows a hybridization result comparison between using microarray agitation according to the present invention in a water bath and traditional microarray incubation without agitation in a water bath as an experimental control. Otherwise, experimental conditions were identical: (i) identical biological samples, (ii) identical probes, (iii) identical hybridization conditions including use of the coverslip approach, hybridization temperature, hybridization time and so on, (iv) identical washing conditions, and (v) identical fluorescent scanner settings.  FIG. 7   a  is a hybridization scan of a DNA microarray incubated overnight in a water bath using microarray agitation according to the present invention.  FIG. 7   b  is a hybridization scan with the same parameters except using the traditional still (no agitation) incubation method as an experimental control.  FIG. 7   c  is a detail of the upper left hand corner of  FIG. 7   a .  FIG. 7   d  is a detail of the upper left hand corner of  FIG. 7   b . It is observed that the microarray ( FIGS. 7   a  and  7   c ) incubated with agitation by the present invention results in substantially improved hybridization signal intensity and uniformity, compared with the microarray ( FIGS. 7   b  and  7   d ) incubated under control conditions. The improvement may be due, in at least part, to enhanced fluid transport of the hybridization buffer under the coverslip caused by microarray agitation with the present invention.  
         [0029]     The present invention can be implemented in disease diagnostic, biological and agricultural research, food safety detection, forensic authentication and their related fields.  
         [0030]     Variations and extensions of the embodiments described are apparent to one of ordinary skill in the art. For example, in reference to  FIG. 5 , a holder to affix microarray  114  to sample plate  109  could be used to prevent microarray  114  from slipping off sample plate  109 . Also, embodiments of the invention can be used to mechanically agitate devices or samples other than microarrays. Other applications, features, and advantages of this invention will be apparent to one of ordinary skill in the art who studies this invention disclosure. Therefore the scope of this invention is to be limited only by the following claims.