Abstract:
A system for dispensing microdrops of reagent in small, precisely metered quantities maintains the fluid reagent to be dispensed in a reservoir under a controlled pressure. The reagent is dispensed through multiple nozzles connected to solenoid-actuated valves that control the flow of the reagent from the reservoir to the nozzles. Each valve is connected to one of the nozzles and electrical pulses are supplied separately to each of the valves to separately control the opening and closing of each valve to dispense predetermined quantities of the reagent through each nozzle at predetermined times.

Description:
PRIORITY CLAIM 
       [0001]    The present application is a continuation of U.S. application Ser. No. 10/347,085 filed Jan. 17, 2003, which claims priority from U.S. Provisional Application No. 60/351,700 filed Jan. 24, 2002, and entitled “PRECISION LIQUID DISPENSING SYSTEM.” 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    The invention relates generally to systems for depositing small amounts of liquid having volumes in the range of about 0.5 μL to 2 mL. Although there are various end uses for such systems, they are particularly useful in connection with microscale chemical and biological analyses. Frequently, the microdispensing system will be used to dispense reagent into a microplate having an array of small wells which hold liquid. A common size is a 96 well plate, measuring about 80 by 120 mm and having round sample wells having a diameter of about 6.5 mm. More recently, plates having 384 and 1536 wells have become available, and the wells in such plates are correspondingly smaller. Thus, reagents must be dispensed in extremely small quantities, and achieving dispensing accuracy and repeatability becomes increasingly difficult. 
         [0003]    Depositing small droplets of liquid for various purposes, including ink jet printing has been of interest in recent years. For example, in U.S. Pat. No. 5,743,960 a system using a solenoid valve is employed. The system features the use of a positive displacement pump to provide the needed flow while the solenoid valve is opened and closed to form the desired droplet size, said to be in the range of 1-4 nanoliters (1-4 mL). Substitution of a piezoelectric dispenser for the solenoid valve dispenser was suggested. The volume of liquid deposited was intended to be in the range of 0.42×10 −9  to 2×10 −6  liters (0.42 mL to 2 μL). 
         [0004]    Another patent disclosing the use of a positive displacement pump to supply a piezoelectric dispensing nozzle is U.S. Pat. No. 6,203,759. In an alternative system, a reservoir containing a liquid is maintained at a desired pressure. In both types of dispensing systems a sample typically is aspirated into the piezoelectric nozzle and then dispensed, using a liquid different from that being dispensed. 
       SUMMARY OF THE INVENTION 
       [0005]    In accordance with the present invention, a system is provided for dispensing microdrops of reagent in small, precisely metered quantities from a reagent reservoir containing a fluid reagent to be dispensed. The reagent in the reservoir is maintained under a controlled pressure, and is supplied to multiple nozzles for dispensing microdrops of the reagent. Multiple solenoid-actuated valves are connected between the reservoir and the nozzles for controlling the flow of the reagent from the reservoir to the nozzles, with each valve being connected to one of the nozzles. Electrical pulses are supplied separately to each of the solenoid valves to separately control the opening and closing of each valve to dispense predetermined quantities of the reagent through each nozzle at predetermined times. 
         [0006]    In a preferred embodiment of the invention, the reagent in the reservoir is maintained under a controlled pressure by an air pump that supplies pressurized air to the reservoir. An electrical control signal is supplied to the pump to control the pressure of the air supplied by the pump to the reservoir. A transducer senses the pressure within the reservoir and produces a signal representing that pressure. A closed-loop control system uses the transducer signal in a PID algorithm to maintain the desired pressure in the reservoir by regulating the electrical control signal supplied to the pump. 
         [0007]    A preferred arrangement for controlling the solenoid valves permits selection of the desired volume of reagent to be dispensed from each nozzle, and a calibration table that specifies the widths of the electrical pulses required to dispense specified volumes of the reagent. When a desired volume not specified in the table is selected, a required pulse width is calculated from the pulse widths specified in the table for the two specified volumes closest to the selected desired volume. The calculation is preferably performed using linear interpolation between the two closest values in the table. 
         [0008]    The invention provides improved accuracy and repeatability of dispensing, with the option of dispensing smaller volumes not currently available in commercial products utilizing solenoid valves. The invention allows dispensing in low volumes, e.g., from 0.5 μL to 2 mL, with full chemical compatibility with common chemical reagents used in biotechnology and chemical laboratories. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0009]    The invention may best be understood by reference to the following description taken in conjunction with the accompanying drawings, in which: 
           [0010]      FIG. 1  is a block diagram of an eight-nozzle precision reagent-dispensing system embodying the invention; and 
           [0011]      FIG. 2  is a block diagram of a 32-nozzle precision reagent-dispensing system embodying the invention. 
       
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
       [0012]    Although the invention will be described in connection with certain preferred embodiments, it will be understood that the invention is not limited to those particular embodiments. On the contrary, the invention is intended to include all alternatives, modifications and equivalent arrangements as may be included within the spirit and scope of the invention as defined by the appended claims. 
         [0013]    Turning now to the drawings, and referring first to  FIG. 1 , the reagent to be dispensed is contained in a reservoir  10  (preferably a glass container) having a pressurized headspace at the top of the reservoir. An output line  11  leads from a supply line  12  near the bottom of the reservoir  10  to a manifold  13  having eight output lines  14   a - 14   h  leading to eight high-speed, solenoid-actuated valves  15   a - 15   h . Each valve  15  carries a dispensing nozzle  16 . Whenever one or more of the valves  15  is open, the pressure in the reservoir  10  forces reagent from the reservoir through the line  11  and the manifold  13 . Manifold  13  is designed to allow equal flow distribution from the single output line  11  to the eight output line  14   a - h  and to the open valve(s)  15  is to the corresponding dispensing nozzle(s)  16 . In a preferred embodiment, the manifold  13  is equipped with a bottom seal fitting  13   a , has a fully swept internal liquid path to reduce the possibility of trapping air and is made of a polyaryletherketone (“PEEK”) resin which provides good mechanical properties in combination with good resistance to the types of reagents commonly used in this type of equipment. The line  11  leading to the manifold  13  is preferably 0.125″ ID, 0.1875″ OD PFA Teflon® tubing, and the lines  14   a - 14   h  connecting the manifold  13  to the valves  15   a - 15   h  are preferably 0.040″ ID, 0.0625″ OD Tefzel® tubing. Lines  14   a - 14   h  and line  11  are coupled to the internals of manifold  13  in a manner that avoids unequal flow distribution, additional restrictions in metering, or trapping of air, all of which cause degradation of target dispense accuracy and precision. 
         [0014]    The pressure within the reservoir  10  is controlled by an air pump  20  that supplies pressurized air to the reservoir via line  21  at a controlled pressure, e.g., about 5 psig (34.5 kPa gauge). The pump  20  preferably includes a brushless DC motor (with a three-wire control option) that is controlled by a system controller  22  via electrical line  23 . The system controller  22  includes a microprocessor that receives a feedback signal from a transducer  24  sensing the pressure within the reservoir  10 . The transducer  24  is connected to a pressure tap line  25  that comes off of the reservoir  10 , and generates an electrical signal on line  26  corresponding to the pressure sensed by the transducer in the tap line  25 . The pressure supply line  21 , the pressure sensor tap line  25 , and the reagent supply line  11  enter/exit the reagent reservoir  10  through a cap  10   a . The lines  21  and  25  are attached to the cap  10   a  via barb fittings, while the liquid supply line  11  passes through the cap and is captured by a flangeless fitting  11   a . The lines  21  and  25  are preferably 0.125″ ID, 0.25″ OD Tygon® tubing. 
         [0015]    The microprocessor in the system controller  22  uses the signal from the transducer  24  in a standard PID (proportional, integral, derivative) control algorithm, to produce an output signal on line  23  to control the pressure within the reagent reservoir, preferably to within 0.02 psi. That is, the microprocessor continually compares the actual reservoir pressure, represented by the transducer signal on line  26 , with the desired or “set point” pressure, e.g., 5 psig (34.5 kPa gauge), using the PID algorithm to produce the requisite output signal for maintaining the desired pressure in the reservoir  10 . The pressure is maintained within a variation of about ±0.5%. A preferred minimum flow rate for the pump  20  is 500 ml/min. at a pressure of 5 psig. 
         [0016]    The system controller  22  also produces the electrical pulses that control the times at which each of the valves  15   a - 15   h  is opened and closed. These pulses are generated on any of eight different output lines  30   a - 30   h , each of which is connected to one of the solenoid-actuated valves  15   a - 15   h . Each pulse rises from a differential voltage of zero to 24 DC volts spike pulse for 2 milliseconds, then reduces to a differential of 5 volts to hold open the valve  15  receiving that pulse, remains at the 5-volt level for a time period sufficient to dispense the selected volume of reagent through the opened valve, and then drops to a differential voltage of zero volts at the end of that time period to close the valve. 
         [0017]    The desired volume of reagent to be dispensed from each nozzle  16  is selected by the user via a keypad or other manual input device on the front of a control panel (not shown). This manual input provides the microprocessor with a signal representing the selected volume. A memory associated with the microprocessor stores a calibration table that specifies the widths of the electrical pulses required to dispense specified volumes of the reagent. When a volume not specified in the table is selected, the microprocessor calculates a required pulse width from the pulse widths specified in the table for the two specified volumes closest to the selected volume. This calculation is preferably performed using linear interpolation between the two closest values in the table. 
         [0018]    The calibration table is generated initially by supplying one of the solenoid-actuated valves with a succession of pulses of progressively increasing width, and measuring the actual volume of reagent dispensed through the nozzle connected to that valve. These measured volumes are stored in the table, along with the pulse width that produced each volume. Then when the user selects a desired volume, the microprocessor finds either that volume, or the two closest volumes in the table. If the exact value of the selected volume is in the table, the microprocessor generates a pulse having the width specified for that volume in the table. If the exact value is not in the table, then the microprocessor uses the two closest volume values, and their corresponding pulse widths, to calculate the pulse width required to dispense the volume selected by the user. Linear interpolation may be used for the calculation. 
         [0019]    The solenoid-actuated valves used in the dispensing system may be selected on the basis of the specified minimum volume to be dispensed by the system. For example, if the specified minimum volume to be dispensed is 0.5 μL, a valve capable of dispensing a volume of approximately 0.125 μL is preferably selected, to allow for a four to one safety factor. 
         [0020]      FIG. 2  illustrates four dispensing systems of the type illustrated in  FIG. 1  arranged in parallel to provide simultaneous dispensing of reagent from 32 nozzles  40   a - 40   h ,  41   a - 41   h ,  42   a - 42   h  and  43   a - 43   h . This arrangement allows rapid filling of multiple wells in microplates having large numbers of wells. 
         [0021]    In a test of the invention, a sample plate having 96 wells was used, each row of eight wells received sample liquid simultaneously. After each row received samples, the next row of wells received samples of the liquid and so on until all 96 wells had been sampled. Each of the eight valves (Lee Valve Company) opened for 5.0 milliseconds and dispensed 0.5 μL of the sample liquid into a well which had been primed with 199.5 μL of deionized water. After each plate had received 96 samples, the liquid delivery system was flushed and re-primed to simulate commercial practice and thus, to introduce potential variation in the amounts of liquid delivered to each well associated with changing or replenishing the dispensed liquid. 
         [0022]    The liquid dispensed was a 5 g/L solution of a tartrazine yellow dye in deionized water, contained in a 1000 mL bottle, which was pressured to 5 psig ±0.02 (34.5 kPa). The arrangement of the tubing supplying liquid to each valve was made as uniform as possible. Measurement of the amount of liquid dispensed was done indirectly by reading the optical density of the liquid with a Spectracount® photometer (Packard Instrument Company). Values for the ten sample plates are shown in the following table. 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
               
               
                 Plate No. 
                 Mean Optical Density Reading 
                 Coeff. Of Variation, % 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 0.3738 
                 1.23 
               
               
                 2 
                 0.3729 
                 1.42 
               
               
                 3 
                 0.3725 
                 1.32 
               
               
                 4 
                 0.3674 
                 1.62 
               
               
                 5 
                 0.3723 
                 1.44 
               
               
                 6 
                 0.3711 
                 1.32 
               
               
                 7 
                 0.3676 
                 1.61 
               
               
                 8 
                 0.3644 
                 1.69 
               
               
                 9 
                 0.3698 
                 1.50 
               
               
                 10 
                 0.3680 
                 1.48 
               
               
                   
               
             
          
         
       
     
         [0023]    The mean value of the optical density measurements was 0.370 over all the 10 sample plates, with a standard deviation of 0.003 or coefficient of variation of 0.823%. Within individual sample plates, the minimum coefficient of variation was 1.231% on plate  1 , while the maximum coefficient of variation was 1.686% on plate  8 . The total variation from the mean optical density reading was about 1.25% across all the 10 sample plates. It should be evident that the system of the invention is capable of depositing the very precise and repeatable samples of liquids required for the tests typically carried out in such sample plates. 
         [0024]    In one application of the dispensing system, the nozzles are mounted on a moveable support and moved in the Y plane into a location where the nozzle tips are aligned with the well of a microplate into which microdrops of the reagent are dispensed. The microplate is moved by a separate plate holder and displaced horizontally in the X plane. Thus, the nozzles are moved within the Y axis while the microplate that receives the microdrops moves in the X-axis directly and precisely under the nozzles. Alternatively, the nozzles may be stationary and the microplate moved under the nozzles. It is of course possible to move both the nozzles and the microplate for maximum flexibility and speed of operation. 
         [0025]    In practice, it is not desirable to carry out such movements manually, using visual observation by the operator. To assure accuracy in repetitive steps of dispensing reagent into multiple wells, computer control of the movements of the nozzles and/or the microplate generally will be provided. The operator of the apparatus will instruct the instrument via a graphical user interface or by a separately linked computer to carry out a series of movements intended to transfer reagent from the reservoir to the microplate. It will be appreciated that such a sequence of movements may take place in three dimensions, usually called X and Y defining the position in a horizontal plane and Z defining the position in the vertical direction. 
         [0026]    While the present invention has been described with reference to one or more embodiments, those skilled in the art will recognize that many changes may be made there to without departing from the spirit and scope of the present invention. Each of these embodiments and obvious variations thereof are contemplated as falling within the spirit and scope of the claimed invention, which is set forth in the following claims.