Abstract:
An apparatus and method of printing microarrays by ejecting droplets of electrically conducting liquids from wells ( 12 ) onto a substrate ( 21 ) on top of a charged plate ( 20 ) using electrodes ( 18 ) inserted into the wells ( 12 ).

Description:
[0001]     This application claims benefit of U.S. Provisional Patent Application Ser. No. 60/355,962 filed Feb. 12, 2002, entitled Device for Printing Biofluids by Jaan Noolandi. 
     
    
     BACKGROUND  
       [0002]     1. Field of Invention  
         [0003]     This invention relates to an apparatus and method of making microarrays, specifically to a way of ejecting liquid drops of biological fluids directly onto substrates from a fluid container with multiple fluid wells.  
         [0004]     2. Description of Prior Art  
         [0005]     Research protocols and clinical applications in genomics and proteomics depend on the ability to spot thousands of tiny drops of biological fluids on microscope slides and other substrates. An ability to spot microarrays quickly, reliably, and inexpensively is of considerable interest to researchers worldwide and is of significant commercial value.  
         [0006]     Previous research can be divided into (a) current methods of spotting microarrays and (b) work on drop emission from single ejector nozzles. The current methods of spotting in laboratory and commercial environments are (i) are pin-based spotters (ii) photolithographic techniques, (iii) ink-jet print heads, and (iv) focused acoustic beams.  
         [0007]     The contact pin-based spotter is the most common method of spotting in biotechnology laboratories. A robotic arm dips an array of pins into a well plate and the pins are moved to contact a substrate so that each pin leaves a small spot of biological fluid. This type of spotting is based on contact between the pins and substrate so it risks contamination, and the minimum fluid volume for pin dip leads to wastage.  
         [0008]     Photolithographic techniques dominate the pre-made high density gene chip microarray market. However, they require elaborate nanofabrication masking techniques and bulky, expensive production equipment.  
         [0009]     The work on single ejection nozzles does not show how to build a reliable, high throughput, inexpensive ejection device with a plurality of ejector nozzles that can be easily cleaned, protect the biological fluids, and interface with standard laboratory equipment in a modular fashion.  
         [0010]     The present invention overcomes the limitations above. None of the previous efforts in this field disclose all of the benefits of the present invention, nor does the prior art teach or suggest all of the elements of the present invention.  
       OBJECTS AND ADVANTAGES  
       [0011]     An object of the present invention is to attain high throughput and inexpensive biological fluid handling by simultaneously ejecting drops of biological fluids from a container with many wells directly onto a substrate.  
         [0012]     A further object of the present invention is to eliminate the intermediate step of moving the biological fluids from storage containers into ejection wells.  
         [0013]     It is another object of the present invention to use containers which have wells conforming to but not limited to the established biotechnology industry standards, with 96, 384, or 1536 wells in electrically insulating well plates. The containers can be inexpensively made, discarded when empty, or cleaned and refilled with biological fluids, and conform in a modular fashion for use with automatic fluid handling systems.  
         [0014]     It is another object of the present invention to increase reliability of spotting by covering the openings on both sides of the well plate with inexpensive protective covers to avoid spillage, evaporation, and contamination of the biological fluids before drop ejection. The use of membranes that are punctured by the electrodes during spotting also decreases the evaporation rate as well as contamination by leaving the openings effectively covered.  
         [0015]     It is also an object of the present invention to provide a simple, robust, and reliable method and apparatus for ejecting biological fluids in wells of a well plate onto a substrate.  
       SUMMARY  
       [0016]     The apparatus of the present invention comprises: a non-conducting well plate modified by forming openings in the bottoms of the wells, which wells can each be filled with a different electrically conducting biological fluid; an array of conducting electrodes that can be dipped into the wells; a substrate located below the openings which is positioned on an electrical ground plane; and a power supply capable of applying electrical potential pulses to one or more electrodes at a time to form electric fields that cause the wells eject drops onto the substrate.  
         [0017]     The method of the present invention consists of ejecting drops of electrically conducting biological fluids onto a substrate resting on an electrically grounded plate from openings in the wells on the underside of the well plate when voltage pulses from a power supply are applied to the conducting electrodes which have been immersed into the biological fluids within the wells of the well plate. 
     
    
     DRAWINGS  
       [0000]     Drawing Figures  
         [0018]     One example of a method and apparatus according to the present invention will now be described with reference to the accompanying drawings in which:  
         [0019]      FIG. 1  illustrates, diagrammatically, a view of the apparatus of the present invention.  
         [0020]      FIG. 2  shows a modified 384-well plate for storing fluids.  
         [0021]      FIG. 3  shows part of the apparatus of the present invention, in which the holes underneath the well plate connecting to the wells on the other side of the well plate are displayed.  
         [0022]      FIG. 4  shows a sectional view of the conducting pins of the conducting electrode array at the top immersed into the fluids inside the wells of the well plate at the bottom.  
         [0023]      FIG. 5  shows a detail of a conducting electrode immersed into the fluid of a well plate, with an opening connecting to the outside, above a ground plane covered by a substrate.  
         [0024]      FIG. 6  shows the apparatus of the present invention, in which the wells of the well plate are uncovered. 
     
    
     REFERENCE NUMERALS IN DRAWINGS  
       [0000]    
       
           9  Cable leading to power supply  
           10  Device for printing biofluids  
           11  Well plate  
           12  Wells in well plate  
           13  Cover over wells in well plate  
           14  Opening on well plate connecting to biological fluid well  
           15  Channel connecting well in well plate to underside of well plate  
           16  Cover over holes on underside of well plate  
           17  Conducting electrode array  
           18  Conducting electrode  
           19  Molded plastic support for conducting electrode array  
           20  Electrical ground plane  
           21  Substrate covering ground plane  
           22  Biological fluid in well of well plate  
           23  Air gap between hole in bottom of well plate and substrate  
       
     
       DETAILED DESCRIPTION  
       [0040]      FIG. 1  shows the apparatus of the present invention, a device used to eject biological fluids onto a substrate, generally indicated by  10 . The system  10  is connected by the cable  9  to a high voltage pulse generator. The bottom component  11  is a 384 well plate, shown in  FIG. 2 , which can be made out of molded plastic. Each of the wells  12  shown in  FIG. 2  can be filled with a different biological fluid in aqueous solution. After filling each of the wells  12  with a fluid, a cover  13  in  FIG. 1  can be attached over the surface of the well plate  11  to seal the wells  12  and to prevent leakage or evaporation. Typically each well  12  in the well plate  11  accommodates from 1 to 200 microliters of biological fluid. In the preferred embodiment 30 microliters of biological fluid are used. The cover  13  can be a piece of plastic or a membrane, for example 3M brand Scotch Tape. As shown in  FIG. 3 , each of the wells  12  also has a hole  14  preformed from the bottom of each well  12  through the plate material to the other side, providing a channel  15 , shown in  FIGS. 4 and 5 , for the fluid to leave the well  12  by as described below. The hole  14  at the bottom of each well has a diameter of 120 microns in the preferred embodiment, but can range from 10 microns to 300 microns. The underside of the well plate  11  has a cover  16 , typically an adhesive membrane, shown in  FIG. 2 , which can be removed. The adhesive membrane can be 3M brand Scotch Tape.  
         [0041]      FIG. 1  also shows the conducting electrode array  17 , consisting of 384 conducting electrodes  18  embedded in molded plastic  19 , such that the conducting electrode array  17  fits over the well plate  11 , with the conducting electrodes  18  inserted into each of the wells  12 . In other embodiments, the wells  12  can range in number from 2 to 10,000. In the preferred embodiment, the number of electrodes  18  matches the number of wells. In another embodiment there can be fewer electrodes than wells. In the preferred embodiment, the conducting electrodes are stainless steel pins. In other embodiments any non-reactive conductor may be used. When the conducting electrode array  17  is aligned with the well plate  11 , the conducting electrodes  18  can be pushed through the membrane  13  covering the wells  12 . This leaves the wells  12  covered and minimizes evaporation and contamination. Each of the conducting electrodes  18  in the conducting electrode array  17  is connected to power supply that can supply 500 volts to 4,000 volts, with 3,000 volts in the preferred embodiment. The voltage can be pulsed from 0.2 milliseconds to 20 milliseconds, and is pulsed for 2 milliseconds in the preferred embodiment. In another embodiment, the electrodes are embedded in the material forming the wells  12  and these are connected to the power supply.  
         [0042]     The conducting plate  20  is shown in  FIG. 5 , covered by a substrate  21  which can be but is not limited to glass, nitrocellulose, and nylon, onto which drops of biological fluid  22  are ejected from the wells  12  of the well plate  11 .  
         [0000]     Operation of Invention  
         [0043]     The process of printing biological fluids  22 , shown in  FIG. 5 , onto the substrate  21  is described as follows: the adhesive tape  16  (shown in  FIG. 2 ) covering the underside of the well plate  11  (containing pre-filled biological fluids  22 ) is removed. The well plate  11  is positioned as shown in  FIG. 1  above the substrate. The conducting electrodes  18  of the conducting electrode array  17  are positioned over the well plate  11 , as shown in  FIG. 1 , lowered to puncture the membrane  13  covering the wells  12 , and immersed into the biological fluid  22  (see  FIG. 5 ) in each well  12 . The device for printing biofluids  10  of  FIG. 1  is positioned above a substrate  21  (see  FIG. 5 ) covering a conducting plane  20 , with a gap from the opening  14  on the bottom of the well plate to the ground plane  20  of 50 microns to 1,000 microns, as shown in  FIG. 5 , with 400 microns in the preferred embodiment. The air gap from the opening  14  on the bottom of the well plate  11  to the substrate  21  can be in the range 30 microns to 900 microns, with 250 microns in the preferred embodiment. An electrical pulse is selectively transmitted through the conducting electrodes  18  into the biological fluids  22 , which are also conducting since they consist of aqueous ionic solutions of biological entities.  FIG. 5  shows an arrangement for one of the wells  12  in the well plate  11 .  
         [0044]     The configuration for spotting a 20 microliter volume of plasmid DNA (12 kiobases) in 10 mM Tris-Acetate buffer pH 8.2 onto a nitrocellulose substrate can be: nozzle interior diameter of 120 microns, a gap from nozzle to ground plane of 400 microns, and a 3,000 Volt electrical pulse for 2 milliseconds duration.  
         [0045]     A number of strategies can be used for applying voltage pulses to achieve drop ejection onto the substrate. The electrical potential pulse can be an oscillating voltage, which causes ejection of a biological fluid from a well, or the oscillating voltage causing drop ejection can be superimposed on a second voltage which by itself is not enough to cause drop ejection.  
         [0046]     By controlling the electrical pulses on the conducting electrodes  18  in the conducting electrode array  17  in  FIG. 4 , a number of drops can be printed onto a substrate  21 , shown in  FIG. 4 , underneath a stationary conducting electrode array  17 . After one substrate is printed, a new substrate can be put in its place. The printing can thusly proceed to spot many substrates  21  until the well plate  12  is empty, at which point it can be discarded or cleaned and reused. The conducting electrodes  18  in the conducting electrode array  17  can then be washed and reused for printing from another well plate  11 .  
         [0047]     As shown in  FIG. 1 , the substrate  21  can be positioned below the well plate  11 . Since our modified well plate  11  in  FIG. 1  becomes, in effect, a 384 nozzle biological fluid printing device  10 , we are able to print  384  spots from the single well plate  11  onto a substrate  21 , as shown in  FIG. 1 .  
         [0048]      FIG. 6  shows the apparatus of the invention in which the cover  13  covering the well plate  11  shown in  FIG. 1  has been removed.  
       CONCLUSION, RAMIFICATIONS, AND SCOPE  
       [0049]     The present invention is a simple, non-contact, modular printing system with low likelihood of biological fluid contamination and which enables a rapid rate of drop delivery to a substrate. In an embodiment used in a high-performance system, either a single well plate with a few thousand wells or many well plates in a row can all be made to eject simultaneously so that high throughputs can be achieved. Experts predict clinical applications of microarrays will require pharmaceutical companies to produce millions of microarrays. The present invention is capable of economically meeting this need.  
         [0050]     The present invention has important applications in combinatorial chemistry, which is an important research tool for drug design and development. Combinatorial chemistry involves putting many fluids into containers (well plates), carrying out assays and removing the fluids to find the optimal proportions for chemical reactions.  
         [0051]     Biotechnology laboratories and Genome Centers require an easy-to-use and reliable spotting system for monitoring the expression of many genes in parallel, which is provided by the present invention.  
         [0052]     An important application for structural genomics research and structure-based drug discovery is the efficient preparation of protein crystals using nanodroplets. The high-throughput system described in this invention for drop generation and delivery of biological fluids consisting of dissolved proteins to a substrate will facilitate research in this field as well.  
         [0053]     Accordingly, the scope of the present invention should be determined not by the embodiments described above, but by the appended claims and their legal equivalents.