Abstract
Introduction
96-tip pipetting machines have become a popular piece of laboratory equipment for plate cloning, TLC spotting, and a number of other high throughput screening applications where rapid pipetting to a 96-well format is needed. The Robbins Scientific Hydra-96 (Robbins Scientific Corp., Sunnyvale, CA) is a 96-tip pipetter which uses an 8 by 12 array of precision glass syringes 1 . Two syringe sizes are available for dispensing 0.5 – 290 and 1.0 – 580 μL respectively. Teflon coated syringe needles, at lengths to accommodate shallow well and deep well plates, are available. The Hydra can be operated either manually by placing a 96-well plate into the unit and pressing the appropriate front panel aspirate/dispense push-buttons or it can be incorporated into a robotic system where plates are placed into the unit with a robotic arm (i.e. HP ORCA, CRS, Zymark, etc.) and the unit is controlled via its RS-232 port.
In the Hydra-96 design, the syringe barrels are held stationary and a drive motor drives all syringe plungers simultaneously. Plates are front loaded onto a plate elevator which has a second drive motor to move the plate up into the syringe needles. One disadvantage of the Hydra-96 is no needle wash has been provided. To eliminate sample carryover, thorough syringe/needle washing is critical aspect of any pipetting system, especially pipetters which do not use disposable tips. To perform a syringe/needle wash with the Hydra-96, a tray of wash fluid must be placed onto the plate elevator and a number of aspirate/dispense operations performed. The wash reservoir must then be unloaded from the Hydra-96 and discarded. Whether the unit is used manually or with a robotic system, the current wash method is an inefficient use of time and wash reservoir consumables.
To improve syringe/needle wash operations, an automatic needle wash apparatus has been developed and adapted to the Robbins Hydra-96 which permits automatic wash basin filling, positioning, and draining and can be controlled with either front panel switches or remotely from a computer via RS-232. The needle wash apparatus consists of a motor driven wash basin which is automatically positioned up through the Hydra-96 base plate and plate elevator into the syringe needles. The wash basin is automatically filled with a remote pump and drained via a vacuum solenoid. The wash basin has sufficient depth to wash long syringe needles used for deep well pipetting or can be partially filled to wash needles after pipetting operations using shallow well plates.
Design Details
Mechanics
The Hydra-96 is mounted on top of the needle wash apparatus enclosure. A cutout is machined into the base plate and elevator plate of the Hydra-96 which permits the wash basin to be moved up vertically submerging the syringe needles in the wash fluid. The HDPE wash basin can hold sufficient fluid to wet the outside of long syringe needles. An inlet port on the wash basin is used to fill the basin via a remote gear pump. The volume of fluid added to the basin is controlled by cycling the run time of the pump. The basin is drained via an outlet port which is directed to a vacuum bottle and controlled via a solenoid valve.
The wash basin is connected to a telescoping tubing elevator with an integral gear rack. A 12 vdc 128:1 gear motor (Portescap Co.; Hauppauge; part no. 23L21-213E.5/K24.0 128:1) powers a pinion gear coupled to the gear rack which drives the inner telescoping tubing and basin up into position. Upper and lower drive limits are sensed via two reflective optical switches mounted to the basin drive housing. The basin elevator mechanism is placed in an enclosure underneath the Hydra-96.
Electronics
When performing needle wash operations using a remote computer, it was desired to use a single RS-232 connection which communicates to the needle wash station as well as the Hydra-96. To accomplish this task, a MAX333A (Maxim Integrated Products, Inc.; Sunnyvale, CA) analog switch, IC-1, was used to switch the RS-232 connections between the Hydra and the needle wash controller via the RTS hardware control signal from the control computer. A RTS logic HI connects the RS-232 lines to the needle wash controller via the appropriate SPDT connections on the MAX 333A. A logic low signal on the RTS line directs the RS-232 input signals connections to a DB-25 connection, TB1, which is attached to the Hydra-96 remote control input. A +/- 15 vdc dc-dc supply powers the analog switch insuring the analog switch functions at any RS-232C logic level.
The heart of the needle wash controller uses a Micromint (Micromint Inc., Veron CT) Domino-52 single chip microcomputer/controller, uPl, with an embedded BASIC Interpreter. This compact 20 pin IC has a RS-232 port with up to 19.2 kbps, full floating-point BASIC interpreter, 32-KB SRAM, 32-KB EEPROM, 8-bit parallel port, and two ADCs. The unit is programmed using Intel's Basic 8052 command set and custom assembly language CALL routines supplied from Micromint. To power the 12 vdc gear motor, an H-bridge FET driver, FET1–4, was used. Appropriate logic levels from ports P10 and P11 cause the H-bridge to supply 12 vdc to the motor in a forward, reverse, or off mode. To sense the location of the basin, opto sensors, OPT-1,2, are used. The sensors are mounted to the drive mechanism and operate in a reflective mode where OPT-1 senses basin up and OPT-2 basin down through ports P14 and P15.
A 12 vdc gear pump (Cole Parmer; Niles, Ill; cat. no. H-07012-20) is used to fill the basin. The power to the pump is switched using port P16 of the microcomputer and an Opto-22 (Opto-22; Temecula, CA) opto-isolated dc output module, MOD2. The module can accommodate a current up to 3 amps. To limit the flow rate from the pump, a power resistor, R12, is placed in the return lead of the motor. A pump voltage of 8 volts is sufficient for proper priming of the gear pump and a flow rate appropriate for the basin.
A 12 vdc solenoid valve (Humprey; Kalamazoom MI; part no. 32012) is used to drain the basin via port P17. An Opto-22 dc output module, MODI, is used to provide the necessary drive and isolation.
In addition to remote control via the RS-232 connection, the needle wash apparatus may be operated using manual front panel switches. Switch SW1 selects remote or local control, switch SW2 is used to fill/drain the basin, and switch SW3 used move the wash basin up/down. Switches connect to ports ADC0, P13, and P12 respectively. The electronics has been designed onto a custom 4″ × 7″ printed circuit board which mounts inside the enclosure. The enclosure also contains a 12 vdc 1.5 amp power supply which is used to power the controller, pump, solenoid valve, and basin drive motor. Regulator, VR-1, regulates the 12 vdc down to 5 vdc necessary for the embedded controller and TTL logic.
Controller Software
The control computer was programmed using Micromint's editor and Intel's 8052 BASIC command set. The remote control software consists of routines which interpret ASCII commands sent from the remote computer and implements the appropriate control function. Table 1 shows the command set.
When a command is passed to the needle wash controller, software interprets the command and sends an “OK” message back to the remote computer identifying that the command has been completed. In the manual mode, the embedded controller scans the remote/local switch SW-1 during power up and calls the appropriate manual run routines. To program basin fill time, the first time the fill/drain switch, SW-2, is pressed the fill pump will turn on and run for the duration of the button closure. This time value is stored in memory. The second closure will open the vacuum solenoid for the duration of the closure. Both values are then stored in memory.
Future presses of the push-button cause the pump and vacuum solenoid to run for the appropriate duration in an alternative manner. Pressing the up/down switch, SW-3, causes the elevator to move the basin to the appropriate position. As a safety, in case of power failure, the software will position the wash basin down and drain the basin for 15 seconds after a new power up.
Conclusion
A needle wash station has been designed and adapted to the Robbins Scientific Hydra-96 pipetter which permits automated needle wash operations without having to shuttle wash reservoirs in and out the Hydra-96 manually.
Needle Wash Station Remote Control Command Set
A wash basin moves up into the wash needles and will fill and drain automatically. Sufficient wash fluid accommodates long syringe needles used for deep well plates.
The needle wash station can be easily integrated into a totally automated robotic screening system and controlled via a simple ASCII command set through a single RS-232 connection. The needle wash station is modular and can be easily adapted to other pipetting machines.