Anal. Chem. 1998, 70, 5085-5092
Technical Notes
System for Preparing Microhybridization Arrays on Glass Slides David J. Graves,*,† Hung-Ju Su,†,‡ Steven E. McKenzie,†,§ Saul Surrey,§ and Paolo Fortina*,‡
Department of Chemical Engineering, University of Pennsylvania School of Engineering and Applied Science, Philadelphia, Pennsylvania 19104, Department of Pediatrics, University of Pennsylvania School of Medicine and The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, Department of Pediatrics, Jefferson Medical College, Philadelphia, Pennsylvania 19107, and the du Pont Hospital for Children, Wilmington, Delaware, 19803
We describe the construction and operation of an arrayer system to produce patterns of DNA sequences for analytical uses such as microarrays of oligonucleotide on microchips. Detailed documentation on construction is provided, as well as added electronic circuitry and the software for the instrument, including programs to machine its own working surface as well as those to operate it as an arrayer. Its cost is modest, and with a single droplet tip it can deposit 96 spots per slide on 32 slides in about 200 min (readily upgraded to higher speeds). As currently operated, it can place 400 spots in 1 cm2, and this density, too, can be increased easily. We discuss design features and performance to demonstrate utility and flexibility. An interesting and useful recent development in analytical biotechnology is the microarray. Any number from roughly a few dozen to several hundred thousand different spots of cloned DNA, oligonucleotide, etc. (probes) are created at known locations on a glass or other planar substrate. The probes can be either attached to the surface or synthesized in place. These can then be used to identify and quantify large numbers of fluorescently tagged DNA or RNA (targets) simultaneously because of the highly specific hydrogen bonds that form between complementary bases. The theory and applications of this technology are well known and will not be discussed here.1-8 * Address correspondence to David J. Graves, Sc.D., Department of Chemical Engineering, University of Pennsylvania, School of Engineering and Applied Science, 220 S. 33rd St., 311A Towne Building, Philadelphia, PA 19104. Phone: (215) 898-7951. Fax: (215) 573-2093. E-mail:
[email protected]. Address reprint requests to Paolo Fortina, M.D., The Children’s Hospital of Philadelphia, 310-C Abramson Research Center, University of Pennsylvania, School of Medicine, Philadelphia, PA 19104. Phone: (215) 590-3318. Fax: (215) 590-3660. E-mail:
[email protected]. † University of Pennsylvania School of Engineering and Applied Science. ‡ University of Pennsylvania School of Medicine and The Children’s Hospital of Philadelphia. § Jefferson Medical College and the du Pont Hospital for Children. (1) Chetverin, A. B.; Kramer, F. R. Bio/Technology 1994, 12, 1093-1099. (2) Goffeau, A. Nature 1997, 385, 202-203. (3) Lockhart, D. J.; Dong, H.; Byrne, M. C.; Follettie, M. T.; Gallo, M. V.; Chee, M. S.; Mittmann, M.; Wang, C.; Kobayashi, M.; Horton, H.; Brown, E. L. Nat. Biotechnol. 1996, 14, 1675-1680. (4) Mirzabekov, A. D. Trends Biotechnol. 1994, 12, 27-36. 10.1021/ac980456n CCC: $15.00 Published on Web 10/24/1998
© 1998 American Chemical Society
Table 1. Integrated Circuits Used in Auxiliary Circuits ingegrated circuit
type
A B,J C,D E,F G H I
ICL 7660 (volt converter) CD4053 (3 × 2-channel switch) CD4052 (4-channel mux) CA3130 (operational amplifier) DM7400 (quad nand gate) CD4049 (hex inverter) DM7475 (4× latch)
We describe a system for depositing controlled volume microdroplets on a series of glass slides in precise locations. In addition to our specific application, such a device has other potential uses, such as spotting TLC plates, sampling and diluting liquids in microtiter plates for various applications, automating peptide or oligonucleotide synthesis without a dedicated synthesizer, carrying out various operations in combinatorial chemistry, etc. Although the system is not inexpensive (roughly $15 000), it is considerably less costly than comparable commercial robotic systems and has the precision to generate microarrays, which many commercial systems cannot do very well. Furthermore, the cost could be reduced further by using a smaller X,Y table (currently almost half of the total cost) and/or by using a “fountain pen” type of capillary deposition system favored by some,5 rather than the micropump that we have chosen. Our goal was to produce a reliable deposition system with minimal labor in addition to minimal cost, so we chose a number of “off-the-shelf” items (see Tables 1 and 2) that were easy to interconnect and assemble. The design was guided significantly by the work of Brown and co-workers, who have published a Web page outlining the features of their arrayer (http://cmgm. stanford.edu/pbrown/). Our design differs from theirs in two major ways. First, we used an X,Y robotic table to move an (5) Schena, M.; Shalon, D.; Davis, R. W.; Brown, P. O. Science 1995, 270, 467-470. (6) Southern, E. M. Trends Genet. 1996, 12, 110-115. (7) Yershov, G.; Barsky, B.; Belgovskiy, A.; Kirillov, E.; Kreindlin, E.; Ivanov, I.; Parinov, S.; Guschin, S.; Drobishev, A.; Dubiley, S.; Mirzabekov, A. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4913-4918. (8) Stipp, D. Fortune 1997, 135, 56-73.
Analytical Chemistry, Vol. 70, No. 23, December 1, 1998 5085
Table 2. Microarrayer System Equipment List arrayer component
supplier
14-in. × 14-in. X,Y stage, 10 tpi leadscrew, precision grade 2-in. Z carriage, 10 tpi leadscrew, commercial grade 21-in. × 21-in. × 3/4-in. Plexiglas 24-in. × 42-in. optical breadboard 2.25-in. × 1.5-in. angle iron, 20 ft. length, cut as needed indexer personal computer stepper motor drive units (×3) software to machine working surfaces of arrayer and to operate arrayer flexible shaft tool added circuitry joystick Micro-4 pump controller and UltraMicroPump 5- or 1-µL syringe video monitor video camera video microscope; VZM model 300 video microscope; mounting accessories
New England Affiliated Technologies (NEAT) NEAT local supplier Newport Arrow Star Parker Compumotor any suitable manufacturer Parker Compumotor available on request from authors Foredom contact authors Radio Shack World Precision Instruments World Precision Instruments H&R Co. H&R Co. Edmund Scientific Co. Edmund Scientific Co.
assembly of microscope slides under a fixed needle carrier, which moves in the Z direction. Brown’s arrayer has one axis (Y) for the tray of slides and a second, cantilevered axis at right angles to the first, carrying the droplet deposition system (X,Z). The tradeoffs between the two designs are a somewhat sturdier and simpler system in our case versus a considerably larger total working area in theirs. Also, our Z axis deposition system, which makes only minor vertical excursions, permits bulky deposition devices such as one or more micropumps, piezo deposition nozzles, a video camera monitoring system, etc. to be added with little difficulty. Many of these types of items can be mounted in permanent locations on a fixed overhead rail near the Z motion carriage. Our video microscope is aimed at the surface where the deposition needle touches down. Such bulky items are likely to be too heavy for an X,Z unit that moves rapidly in two dimensions. A moving microvideo camera also might vibrate too much to permit good viewing of the droplet deposition process. Finally, our system requires considerably less machine shop labor to construct than that described by Brown. Nevertheless, both designs are comparable in price and are capable of producing similar arrays. EXPERIMENTAL SECTION Overview of the Arrayer. A schematic of the system is shown in Figure 1. The Parker Compumotor 6200 Indexer has many desirable features and was chosen to control the motors. It has two serial input/output communication ports, one of which can interface to the operator’s computer while the other signals the pump controller. It has inputs for two sets of limit switches, a joystick, and “kill motion” control. The outputs include 24 singlebit inputs, 24 outputs, and microstepping signals to control two motors. Most important, it has a very good macro language built in. This permits the user to specify, for example, required movement in two directions. The indexer then supplies motor signals to accelerate and decelerate both axes simultaneously to arrive at the desired final location without overshoot or missed steps. Such things as number of microsteps per revolution, acceleration and decelearation rates, delay times, error response, motion, etc. can be specified through more than 500 commands. 5086 Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
stock no. OFL-2121-SM RM-200
6200 Digiplan series MM with #44 handpiece UMC4, UMP 15863 or 15862 G3-010 G3-054 P39708 P53885, P53886 (×2), P53889
Commands can be entered through a terminal or with an attached computer. Programs consisting of a series of high-level commands are stored within the indexer in nonvolatile memory. However, individual lines in a given program cannot be edited, and on rare occasions the indexer memory can become corrupted. This means that the whole program must be downloaded each time one wishes to make a change or when the indexer has “forgotten” what to do. For this reason, the programs are written in text form on any available inexpensive computer where they can be edited, printed, saved, and finally downloaded to the indexer. For these reasons, and since it is hardly more expensive than a simple terminal, a computer was selected for operator input. The indexer is relatively expensive. Therefore, the addition of a second indexer just to control the third axis of movement was undesirable. Instead, circuitry was devised to switch or multiplex one of the indexer outputs (Y) between Y and Z control as needed under manual or computer control. Several other circuit functions will be described later. Three Parker Digiplan drive units (D in Figure 1) receive step and direction signals from the indexer and power the three axis motors. A manual joystick control permits one to “teach” the machine approximate locations for a series of movements, which later can be slightly corrected if necessary. The micropump is used to pick up oligonucleotide solutions and deposit them as microdrops on glass slides. Mechanical Features of the Design. The heart of the system is a large X,Y stage moved by robotically controlled stepper motors. Ours has a 21-in. × 21-in. footprint and can move 14 in. in each of the two perpendicular directions (OFL-2121-SM, precision grade, New England Affiliated Technologies). A 3/4in.-thick polymethyl methacrylate (Plexiglas) sheet is bolted to it to form the work surface. The Z axis (vertical) linear movement carriage (RM-200, commercial grade, NEAT) has a 2-in. range of motion. All three axes have 10 tpi leadscrews, with a specified maximum error of approximately 0.0014 in. (35 µm) in the X and Y axes and 0.001 in. (25 µm) in the Z axis. Within the 14-in. × 14-in. center of the main stage, we were able to position eight rows of grooves, each of which can accommodate four standard 1-in. × 3-in. microscope slides (see Figure 2). In addition, two
Figure 1. Overall signal flow. A standard computer stores programs and downloads them to the indexer through a serial port. A second serial port in the indexer controls the drop formation pump. Single-bit signals from an I/O port are fed to a set of auxiliary circuitry to multiplex the Y and Z control and limit signals, process the joystick and switch signals, and invert the limit signals. The X signals go directly to a motor drive unit. The items marked D are motor drive units.
Figure 2. Robotic working area. A flexible shaft tool was mounted temporarily on the Z axis to mill out the various regions on a 3/4-in. Plexiglas sheet. The tip washing station was similarly milled from a 1-in.-thick block that was fixed with removable stainless steel pins to the surface. Retaining walls and clamps for multiwell plates were fixed in milled grooves and holes. Similar pins were used to mount C-shaped stainless steel springs that pressed the microscope slides into reproducible positions in the eight slots. Each slot holds four standard (1 in. × 3 in.) microscope slides.
standard 96-well (or 384-well) microtiter plates and an overflow reservoir for tip rinsing are located within the active area. This design enables us to prepare 32 slides at a time without operator
intervention, each with up to a 768-spot pattern. The number of spots could be multiplied to much higher numbers simply by having the computer pause, signal that new sample plates were needed, and then resume upon an operator response that new plates were in place. The X,Y stage is attached to an optical “breadboard”, and slotted steel angle iron (2.5 in. × 1.5 in., 14 gauge) is used to form a support for the Z axis stepper motor and carriage. It was found that this long horizontal support beam exhibited unacceptable torsional movement under moderate hand presssure even when a doubled angle iron was used. Therefore, additional pieces spaced about 6 in. away from the first pair were added for stiffening (See Figure 3). Although such reinforcement might not have been needed for the small and light droplet deposition system, a key feature of the design was that we intended to have the system act as its own robotically controlled machine shop to form slots, holes, and cavities for the microscope slides, 96-well plates, and tip-washing station. Not only did this reduce our costs, it also guaranteed that the bottom surface of the microscope slide slots was absolutely parallel to the X,Y plane of the moving stage. Added Circuitry. Several electronic circuits were designed to interface the X,Y stage and Z axis motor to the computer. Two of these sets of circuits could be eliminated easily by slight equipment changes, while the third and fourth are more central to the design. Briefly, these circuits are used for providing a set of reference voltages (Figure 4A) and interfacing logic (Figure 4B) for a joystick, a set of inverters to make the NEAT robotic table and the Compumotor controller compatible (Figure 6), a set of circuits to interface pushbuttons and switches for manual input of information (Figure 5), and multiplex circuitry to permit the two-axis controller to control all three axes (Figure 6). Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
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Figure 3. Overview of arrayer. The X,Y stage is mounted on an optical breadboard. The Z axis drive is visible in the center, mounted on standard laboratory construction angle iron. This is also bolted to the breadboard. To the right of the Z drive carriage is the microsyringe pump. Not shown is the video microscope, added later to the left of the Z drive and permanently aimed at the point where the deposition needle contacts the surface. The computer, joystick, switch box, video monitor, and pump controller also are not visible here.
Additional information on the function of these circuits is available from the authors. Droplet Deposition. One signal we use that is not shown on the circuit diagrams is a 9600-baud serial output from the computer. This was intended to interface the indexer with a special LCD controller display for industrial control applications. However, we have used it to control the droplet deposition pump (or pumps). A syringe pump controller from World Precision Instruments (WPI) can control up to four pump heads simultaneously or individually. Although the baud rates and other parameters of the WPI controller and the Compumotor computer are both fixed, they are, fortunately, identical. Thus, one could have a close-spaced array of four deposition needles (for example, a square array on 1-mm centers) that could be filled individually and deposited simultaneously on a slide. All four needles would dip into a single well, but only one pump at a time would withdraw sample. Following a rinse, this would be repeated for the three other wells, filling them one at a time. Alternatively, the deposition needles could be spaced in a 9-mm2 array to conform to the spacing of a standard 96-well plate. They could then be filled and emptied by operating the four pumps simultaneously. This, in effect, would create four smaller arrays simultaneously as is done by Brown and co-workers.5 At present, we use only a single deposition pump and needle, although the system is being upgraded to a four-syringe capacity. Typically, we use a 5-µL syringe for deposition, and the individual droplets are 5 nL in volume and spaced 500 µm (about 0.02 in.) apart. A 1-µL syringe will permit 400-pL droplet formation. The manufacturer claims minimum volume increments about 10 times lower than these, but perhaps because of evaporation, mechanical play, 5088 Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
or surface tension effects, these are the lowest limits that we have been able to achieve reproducibly. “Fountain pen” type capillary tips can be used to produce much smaller droplets. Working Surface and Deposition Tips. Machining the Plexiglas top (see Figure 2) was accomplished by affixing the handpiece of a Foredom flexible shaft power tool to the Z axis carriage. The motor was mounted nearby on a vertical support post with its shaft vertical, producing minimal loading on the Z carriage. A 1/4-in. straight routing bit and a 3/32-in. drill were the only tools needed to create the working surface shown. In addition to the eight slots, each of which was 1 in. × 13 in., we milled deeper holes 1/4 in. in diameter at distances of 1/2 and 1 1/ in. from one end of each slot. This provided recesses for 2 tweezers to remove 3-in. (and custom 1-in.)-long slides from the slots. As the slides are removed one at a time from the right end of the slot, the remaining slides are manually pushed into position over the tweezer well. At the other end of the slots, 3/32-in. holes were drilled, one per slot, to hold short pieces of stainless steel rod. The rod was used as the mounting shaft for a piece of 0.010-in. stainless steel shim stock bent in a C shape, which acted as a retainer spring. These C’s press the microscope slides together, fixing them firmly into position for droplet deposition. At the rear of the slide area, two perpendicular 1/4-in. slots were milled to hold Plexiglas ridges extending about 1/4 in. above the surface of the plastic plate. The springs and trigger mechanisms from two toy water pistols were cemented to the base plate to press the 96-well plates firmly and reproducibly into position against these Plexiglas bumpers. A 2-in. × 2-in. piece of Plexiglas, 1 in. thick, was fixed to the work surface with four stainless steel alignment pins and then machined into a
Figure 4. (a) Sources of voltages for auxiliary circuitry. All circuits are powered by 5 V from the indexer. The 0.9-, 1.0-, 1.1-, and 2.5-V sources are used for joystick slow and fast signals. The -5-V source is used for operational amplifiers in the auxiliary circuits. (b) Joystick speed and direction circuits. One of the two triggers on the joystick chooses low-speed (0.9 and 1.0 V) or high-speed (0 and 2.5 V) signals. A second trigger (see Figure 5) tells the indexer to use these either for a low range or a high range of speeds, providing four speeds in each of the four directions (six with multiplexing). The two outputs from these circuits are X and Y analog input signals.
tip-rinsing bath with inlet and overflow outlet. The pins permit it to be removed for cleaning when desired. The programs we used for all of these milling and drilling operations (as well as operating programs) are available from the authors. The tips used for droplet deposition are cut from 27-gauge (0.016-in. o.d. × 0.008-in. i.d.) stainless steel needle stock. Despite the fact that the Plexiglas tabletop was milled parallel to the movement plane, this tip did not reproducibly touch the surface if it was held rigidly on the Z axis carriage. Therefore, it was suspended in a shorter length of 23-gauge thin-walled stainless steel tubing (0.025 in. × 0.017 in.) which was clamped in place. Connection between the inner tube and the needle of a (nonmoving) 5-µL syringe in the pump mechanism was with 28-gauge (0.013 in. × 0.018 in.) Teflon tubing, long enough for 2 in. of carriage travel, but not excessively long (all tubing and shim stock purchased from Small Parts, Inc., Miami Lakes, FL). The syringe, tubing, and deposition needle must be filled entirely with water and/or deposition solution. Compressible air bubbles, if present, prevent accurate droplet deposition. This Teflon tubing was slightly bowed and clamped about 1 1/ in. above the needle assembly. This causes it to act as a spring, 2 restoring the needle to its lowest position again after it had been lowered into contact with the microscope slide surface and had retracted slightly. With this arrangement, we could ensure that the needle touched the surface on each deposition but that neither the tip nor the slide was damaged. The tip also could be lowered
Figure 5. Computer switch inputs. The top trigger on the joystick provides a velocity range select signal to the indexer. One switch provides an emergency stop function to prevent injury or damage to the arrayer, with a stopped condition signaled by LED. The other four switches provide various manual inputs to control the operation of the indexer.
at a high rate of speed when this spring cushion was in place. The lower end of the needle was cut with a slight bevel rather than exactly at 90°. Otherwise, it tended to seal against the glass surface and interfere with accurate droplet deposition. The general arrangement of this whole needle assembly is visible in Figure 3. The rounded button of the syringe plunger was embedded in epoxy to increase its thickness and provide a flat bearing surface against the WPI pump carriage. This modification improved reproducibility. The Need for Speed. With our present single-needle system, one microscope slide can have 96 spots deposited in approximately 1 h. A full complement of 32 slides takes approximately 3 h and 20 min. These nonlinear times are due to the fact that tip rinsing and sample aspiration take much longer than sequential sample deposition. The time from depositing a previous sample to rinsing and drying the tip is 36 s. From that point to picking up a sample is 12 s. Reaching the first slide takes about 6 s, and subsequent slides require about 2.25 s each. Although this speed will meet our needs for several years, inevitably as we desire to make larger arrays, it will seem far too slow. It should be noted that the modified Brown arrayer design developed by the National Human Genome Research Institute (http://www.nhgri.nih.gov/DIR/LCG/15K/HTML/) describes Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
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Figure 6. Multiplexing and inversion circuits. The six X, Y, and Z limit switch digital signals are inverted to match the indexer input and stage outputs. A temporary-action rocker switch on the joystick is used to permit Z movement rather than Y movement. Either this switch or an indexer digital signal (output 24) controls the multiplexing of the Y and Z signals for limit switches and the stepping signal. Therefore, either Y or Z movement is possible at any point in time, but not both simultaneously.
the preparation of 48 slides with 96 spots in 7 min, compared with the 200 min that ours requires. However, theirs also has 16 tips for simultaneous droplet deposition rather than the one that we currrently use. With their tips, our arrayer could deposit 96 spots on 32 slides in a comparable time, about 12.5 min. Multiple tips are clearly one means to increase speed significantly for large arrays. Our system has size 23 stepping motors with 100 oz. in. of torque for the X and Y axes (and a smaller motor for the Z axis). Although a higher torque motor might have helped speed somewhat, we could have achieved significantly better performance (at considerably higher cost) by choosing linear motors or servo motor systems. The accuracy of spot placement is not a problem with components of the type specified, so one could also trade accuracy for speed by specifying 5 tpi rather than 10 tpi leadscrews. Those who anticipate the need for high speeds because of the number of spots or slides needed may wish to consider these alternatives during the design stages. Another option would be to deposit droplets “on the fly” by using a piezoelectric spraying 5090 Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
system such as that used in ink jet printers. Because motor acceleration and deceleration for each axis take considerable time, the ability to deposit droplets while the carriage is constantly moving would enhance speed dramatically. One need only consider how fast ink jet printers work to appreciate how much of a time savings this could produce. We intend to add such a feature to our system in the future. RESULTS AND DISCUSSION System Performance. The first test, to measure reproducibility of deposition, was conducted with a sample of ink. The scanned image of an array on silanized glass of 400 ink spots, each 5 nL in volume, is shown in Figure 7. Because of unequal evaporation during drying and the resulting segregation of pigment, spot visual density is nonuniform. This is not a problem with surface-reactive oligonucleotides, although images of fluorescently labeled monolayers do show some mottling that apparently is due to uneven surface reaction.9 The uniformity of spacing (9) Sanguedolce, L. A. PhD thesis, The University of Pennsylvania, 1997.
Figure 7. (a) Example of array produced by system. This 20 × 20 array was produced by depositing 5-nL ink droplets at a 500-µm spacing. A smaller deposition tip and/or smaller volumes would permit closer spacing without danger of the droplets running into each other. (b) Example of fluorescently tagged oligonucleotide spots. These 3-fmol spots are on 600-µm centers. Droplets of 5 nL were reacted with a silanized glass surface, and the excess unreacted oligonucleotide was removed by several washing steps. The fluorescent image was obtained with a Storm system.
is excellent (standard deviations of 16 and 27 µm in the X and Y directions, respectively), which includes not only the uncertainty due to mechanical precision but also nonuniform spreading of the droplets on this hydrophobic surface. The difference between these two deviations is due primarily to greater droplet spreading in the Y direction because of the angle and direction at which the needle tip was cut. The lower of these two numbers is probably
close to the inherent mechanical uncertainty of the system. Figure 7b shows spots of a 21-base oligonucleotide whose sequence is included in the β globin gene. It was attached to the surface through an amino group at the 3′ end (spaced from the oligonucleotide sequence by three hexaethylene glycol units).9 The 5′ end has a CY5 fluorescent dye moiety attached to it. Each spot contains approximately 3 femtomoles of oligonucleotide (about three-fourths of a monolayer). This image was obtained on a Molecular Dynamics Storm fluorescence analysis system. Scanning confocal microscope systems provide roughly 10-100 times better sensitivity. The area covered per 5-nL droplet (again, dependent more on surface forces than on mechanical factors) was 1 × 10-3 cm2, with a standard deviation of 1 × 10-4 cm2. The video microscope revealed that, if one attempted to deposit very large droplets, some liquid would adhere to the glass and the remainder to the needle as it was withdrawn, adding significantly to the uncertainty in area quoted above. It was also observed that if the system was paused for a significant period of time (a minute or so), there was enough evaporation in the needle to result in erratic deposition for the first few slides. This can be corrected by manually operating the pump control to expel a droplet, touching it to a hand-held slide, and resuming. One could also arrange for the needle to remain submerged in a solution when the system is paused. Studies were carried out to determine what contamination of a new sample by the previous one could be expected following a rinsing cycle. The deposition needle was filled with 1 µL of a fairly concentrated sodium fluorescein solution (0.5 mg/mL). A row of five droplets was then deposited (25 nL/droplet), and 2 µL of solution (the remainder of the dye solution plus the water behind it) was ejected. Then 2 µL of clean water was drawn up and deposited in a similar row of droplets. This rinsing procedure was repeated four more times. Average signal levels by microscopic fluorometry were 1747, -38, -59, -70, -57, and -35 for the six rows. Although the standard deviations were large (638, 108, 51, 25, 39, and 49) because of the nonuniform pattern of dye deposition, it was clear that two rinses removed substantially all of the dye from the system. Additional tests confirmed that, with three rinses, the dye was undectable (lower limit of detection below 0.1% of the original value). Also of concern was any possible dilution effect by the water in the syringe and Teflon tubing that acts as a “hydraulic fluid” to force out the oligonucleotide solution. Calculations suggest that this will not be a problem. The deposition needle, about 2 in. long, contains approximately 1.5 µL, almost 10 times the volume needed to prepare 32 slides. If the time required to deposit this material were 0.5 h due to some operator interruption (the normal time required is about 2 min), the water would penetrate the solution by diffusion only to a distance of about 1.4 mm. Adding this to the 5.3-mm interface movement due to fluid displacement, one calculates that the water interface and needle tip would still be over 43 mm apart. An experiment to test this prediction was conducted. First, a large volume (2.5 µL) of fluorescein solution was drawn into the syringe. This represents a “worst case”, since this volume would more than fill the needle, creating a mixing volume where it joined the Teflon tubing. The dye was then deposited in very large droplets (100 nL) in four rows of five spots, pausing 10 min Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
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between rows. The total of 2 µL deposited would have been enough to deposit normal 5 nL droplets on 400 slides. The fluorescent signals obtained (again, affected by uneven evaporation patterns) were 1305, 1701, 1957, and 1406 units per row, indicating no observable drop in concentration due to mixing with the water behind the dye solution. A severe test of possible contamination by material remaining from a previous deposition was conducted by drawing up 2.5 µL of dye and then 2.5 µL of water. Five rows of five spots were prepared, again using 100 nL/spot. Average intensity readings for each of the rows were 1.1, -5.7, 2.4, 4.2, and 1302 units. Only after 2 µL of water had been deposited did the dye front become detectable. We conclude that using water behind the oligonucleotide solution to dispense droplets will have negligible potential for diluting them or causing contamination from a previous cycle, provided that an adequate buffer volume is maintained behind the volume to be deposited. This has been borne out by our experience in creating arrays of oligonucleotides for various analyses. Very recently, a commercial arrayer using a similar type of micropump-based droplet deposition system has been described.10
CONCLUSION A relatively simple and economical system can be constructed for reliable preparation of microarrays for genomic and gene expression analysis, combinatorial chemistry, etc. The system has been in use preparing glass slides with oligonucleotide droplets for almost a year with no significant problems of reliability or any failures. The micropump-based needle system described is a viable alternative to the fountain pen type deposition tip described by others. An accompanying video microscope has proved very useful in monitoring droplet deposition and identifying problems rapidly.
(10) Lemmo, A. V. Genet. Eng. News 1998, 18 (14), 30.
AC980456N
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ACKNOWLEDGMENT This work was supported in part by a grant from the Ethel Brown Foerderer Fund for Excellence (P.F. and H.-J.S.), by NIH grants P60-HL38632, P30-HG00425, and R01-DK16691 (D.J.G., P.F., S.S., and S.E.M.), a Special Opportunity Award of the Whitaker Foundation (D.J.G. and S.M), a grant from the University of Pennsylvania Cancer Center (D.J.G.), and by the Nemours Foundation (S.M. and S.S.).
Received for review April 28, 1998. Accepted September 16, 1998.
