The requirements of miniaturized assays and other advanced diagnostics pose technical challenges for the development of reagent dispensing systems.
In recent years, both the diagnostic and pharmaceutical industries have begun to seek improved methods of reagent dispensing that would enable them to reduce the amount of reagent used, improve the accuracy and repeatability of the quantities dispensed, and generally expedite the processing of assays.
The forces driving the search for improved dispensing technologies include the need to increase the amount of diagnostic information that can be derived from a patient sample, and a generalized market pressure to reduce the cost of assays. Characteristics of a successful technology will include the abilities to handle a wide range of solvents and solutes, to dispense reagents in drop volumes in the range of picoliters to microliters, and to increase throughput by using noncontact dispensing methods.
This article discusses the development and characteristics of the Biojet (Bio-Dot; Irvine, CA), a dispensing system that combines a positive-displacement pump with a drop-on-demand solenoid valve to overcome the limitations of traditional dispensing systems.1 The new system is quantitative (dispense volumes can be programmed), has minimal sensitivity to the physical chemistry of the reagents it dispenses, requires no contact or near-contact of the reagent with the target substrate, and is suitable for small-volume delivery in the range from 4.16 to 10,000 nl (see Figure 1).
Current Dispensing Technologies
Traditional methods of dispensing reagents for membrane- based and microwell-based diagnostics include positive-displacement (syringe and rotary piston) pumps, pin arrays, and aerosol systems such as airbrushes. Although considerable research is being conducted to overcome the limitations of these systems, the requirements of miniaturized assays and other advanced diagnostics continue to pose technical challenges for the development of reagent dispensing systems.
Figure 1. An XYX system using two Biojets for dispensing dot and line patterns.
Positive-Displacement Pumps. In general, positive-displacement pumps are programmable and capable of dispensing precise quantities of reagent, but there are some limitations to such systems. To dispense drop volumes less than 3 µl, for instance, they require contact or near-contact of the reagent with the substrate onto which it is being delivered. This requirement increases the time that the system must spend between dispense cycles, thus reducing the speed and throughput of the process. Moreover, to bring the reagent into contact or near-contact with the substrate means also bringing the tip of the dispense element perilously close to it, and this can result in cross-contamination or damage to the substrate. The potential for inflicting surface damage can be particularly important when dispensing onto membranes or other sensitive surfaces. The performance of positive-displacement pump systems can be improved somewhat by using touch-off methods, in which the meniscus of the syringe tip contacts the surface of the target substrate or of liquid in a microwell. This method can dispense drop volumes as low as 500 nl, but the need for contact limits its utility to target substrates that are not easily damaged.
Pin Arrays. The use of pins is common in such applications as the transfer of reagents from one microwell to another, or from a microwell to a membrane. To achieve reasonable throughput, pins are usually formatted in arrays (96- and 384-pin arrays are typical). Generally, each pin dispenses only one drop on each transfer cycle, but many reagent transfers can be done in parallel, thus increasing the speed of the process. Reproducibility of drop size can be good, but volume control depends heavily on the physical chemistry of the reagent being dispensed. Drop volumes down into the picoliter range can be achieved. To prevent any possibility of cross-contamination, pins need to be washed between transfers. The potential for damage to the substrate surface is also an issue that needs to be taken into consideration.
Aerosol Systems. Aerosol systems have been used extensively in biodiagnostic applications for dispensing a wide range of reagents onto absorbent surfaces such as membranes. However, because the airflow associated with aerosol systems can readily redistribute reagents sitting on the surface of a substrate, such systems are generally unusable for dispensing reagent onto nonabsorbent materials. Aerosol systems typically possess a dynamic range (maximum dispense rate/minimum dispense rate) greater than 1000, and can therefore be programmed to dispense a wide range of volumes. When dispensing line formats onto membranes, aerosol systems can achieve volumes as low as 0.1 µl/cm; when dispensing dots of reagent, drop volumes can be as low as 50 nl. A drawback of aerosol systems is that they need to be calibrated frequently, because their flow rates can vary widely according to the system settings and physical chemistry of the reagent being dispensed.
Ink-Jet Printing. Although systems such as those described above are the types most commonly used for the dispensing of reagents in diagnostics applications, considerable research is under way to develop new systems that will meet the needs of advancing diagnostics technologies. One idea that has attracted a great deal of attention is the possibility of adapting ink-jet printing technology to the requirements of reagent dispensing. This is an attractive concept, in part because ink-jet printing is inherently a noncontact technology. By eliminating the need for contact or near-contact with the target substrate, manufacturers can increase dispensing speed and throughput while getting rid of the potential problems of cross-contamination and substrate damage.
The starting point for efforts to adapt ink-jet printing technologies to the challenges of biotechnology applications is the continuous ink-jet printer, such as the ones manufactured by Videojet Systems International (Wood Dale, IL), Domino Amjet, Inc. (Gurnee, IL), and others. This type of ink-jet creates a continuous flow of droplets in a closed-loop format. Printing is accomplished by using electrodes to deflect the droplets, forcing them to impinge on the substrate at the position programmed into the system. Continuous ink-jet printers have been successfully modified for reagent dispensing and shown to be useful for some applications. To be used successfully, however, these systems require that reagents fit a limited physical chemistry profile. Where it has been possible to modify reagents so they can be used, the cost of doing so has proved to be prohibitive for many applications.
The most notable attempts to adapt ink-jet technology are those that have abandoned continuous ink-jet printers in favor of using drop-on-demand technologies. In a drop-on-demand system, the user programs the action of a valve that controls the dispensing function (the demand), in such a way that each actuation of the valve produces a single drop of reagent. Efforts to create a functional drop-on-demand ink-jet system have focused on the use of solenoid and piezoelectric valves, which have been shown to be capable of producing drop sizes in the nanoliter and picoliter range, respectively. In theory, a solenoid drop-on-demand valve can dispense drop sizes over a range from 20 to 2000 nl, using practical valve-opening frequencies up to about 500 Hz.2 So far, however, no one has managed to create a commercially viable drop-on-demand dispense system using only a solenoid valve. Piezoelectric valves can produce drop sizes in the range of 100—1000 pl, again with practical valve-opening frequencies up to about 500 Hz. Some success has been achieved in creating a drop-on-demand piezoelectric-valve system, but doing so has required the use of a pressure control system and degassed reagents.3
The major problem with both of these systems is that they are extremely sensitive to the physical chemistry of the reagent being dispensed, and particularly to the effects of dissolved air in the dispensing line. The systems work fine as printers, because the printing inks used have been specifically optimized to work with the systems' valves. But to be successfully used for reagent dispensing, the systems must be able to operate reliably with fluids that have a wide range of properties.
The Drawbacks of Current Technologies. Positive-displacement systems such as syringe and rotary piston pumps offer very accurate and repeatable delivery of reagents, but they generally cannot deliver small drop volumes in a noncontact mode, nor can they operate at high frequencies for high-throughput processing. By contrast, noncontact dispensers such as drop-on-demand ink-jet valves and aerosol dispensers are very sensitive to the physical chemistry of the reagents they deliver—such as surface tension, viscosity, and dissolved air—and are therefore not capable of truly quantitative performance. In addition, such valves are generally pneumatically coupled, and therefore very sensitive to small flow-resistance perturbations in the fluid lines. A major source of such perturbations is bubble formation from dissolved air in the reagents. There is also the inherent problem that most reagents have a significant viscosity-to-temperature coefficient on the order of 1—2%/°C, thus making the dispensing process very sensitive to changes in temperature. Although these valves may exhibit good drop-to-drop repeatability, they must be calibrated frequently to ensure that the desired volume of reagent is being delivered.
A Hybrid Solution
One solution for achieving quantitative noncontact dispensing is a system in which a positive-displacement pump is used to supply reagent to a noncontact dispense element (see Figure 2). Suitable noncontact dispense elements could include piezoelectric displacement, solenoid time-open, orifice, or aerosol units. Such a system combines the quantitative characteristics of a positive-displacement pump with the noncontact ejection characteristics of the selected dispense element. The Biojet system discussed below uses a syringe pump to supply reagent to a solenoid time-open valve, which is used as the dispense element. Much of this discussion is also relevant to other types of noncontact dispense elements when they are configured as part of a positive-displacement system.
Drop Formation. Solenoid dispense elements are commonly used for low-resolution ink printing, where drop sizes in the range of 100 to 300 nl are acceptable. In this system's most common form, the ink or reagent to be delivered is pressurized, and the valve is driven pneumatically. When the valve is opened, a quantity of fluid is ejected through the orifice. The valve can be operated at frequencies up to 1000 Hz with a minimum valve-open time of approximately 200 microseconds. The drop volume of a given reagent is determined by the amount of pressure applied to the reagent, the valve-open time, and the viscosity of the reagent. In this configuration, drop volume is very easily affected by changes in the physical chemistry of the reagent, or by changes in fluid-line resistance.
By replacing the pneumatic valve-opening system with a positive-displacement driver, such as a syringe pump, the system becomes hydraulically coupled (see Figure 2). A positive displacement from the syringe generates pressure at the dispense element; when the element's valve opens, the system delivers a volume of fluid through the valve equal to the positive displacement of the syringe pump. In this configuration, drop volume is determined by the syringe driver, and the valve serves the function of ejecting this volume from the nozzle orifice. Valve-open time can be adjusted to accommodate different drop volumes, but must be at least long enough to permit passage of the displaced fluid through the flow path between the valve seat and the orifice.
Figure. 2 Schematic of a noncontact, positive-displacement dispensing system, showing four different dispense elements that can be used.
A system composed of a positive-displacement syringe and a drop-on-demand ink-jet valve offers important new properties for reagent dispensing. Such a system can be programmed to supply drops of a predictable size, with excellent drop-to-drop repeatability.1 In addition, by using combinations of syringe displacements and high-frequency valve openings, a volume of fluid can be delivered either as a single drop or as a burst of smaller drops (see Figure 3). This capability makes it possible for the system to deliver different drop formats of reagent at a constant volume, which can be useful for automated processing of diagnostic substrates.
The quantitative drop forming process can be put into equation form as follows:
Drop volume = N x M (syringe volume/syringe resolution)
where N is the number of valve actuations, M is the number of motor steps, and syringe resolution is the number of motor steps required for a full stroke of the pump. The ratio of syringe volume to syringe resolution is commonly referred to as the step volume.
The resolution of commercially available syringe pumps such as those manufactured by Cavro (Sunnyvale, CA) or Hamilton Co. (Reno, NV) can range from as little as 3000 to more than 48,000. Typical resolution for such pumps is 24,000, but Bio-Dot has experimentally modified commercial pumps to yield resolutions as high as 192,000.
The above equation can be used to calculate the minimum volume of the drops being dispensed by the system. Since each actuation of the valve produces a single drop (N = 1), the size of each drop is determined by the number of motor steps (M) multiplied by the step volume. If the minimum number of motor steps is used (M = 1), a 50-µl syringe at a resolution of 24,000 steps will produce drops with a volume of 2.08 nl. Increasing the syringe volume also increases the volume of the drop; increasing the syringe resolution decreases the volume of the drop. Thus, if syringe volume is increased to 250 µl and syringe resolution remains at 24,000 steps, the minimum drop volume is 10.4 nl. If the syringe volume remains at 50 µl but syringe resolution is increased to 192,000 steps, the resulting drop volume will be 260 pl.
Varying the number of valve actuations and motor steps can produce useful differences in the type of drops dispensed. Using a 250-µl syringe at a resolution of 24,000 steps, for instance, a single valve opening (N = 1) with 25 motor steps (M = 25) will produce a single drop of 260 nl. By using 25 valve openings (N = 25) and 25 motor steps (M = 25), the same volume of 260 nl can be dispensed as 25 separate drops of 10.4 nl. It is also possible to dispense microliter volumes by using high-frequency bursts of larger drop sizes. For example, using 40 valve openings (N = 40) and 1000 motor steps (M = 1000), the system will deliver 40 drops of 260 nl, for a total volume of 10.4 µl.
The reproducibility of drop size can vary somewhat in this system. For drops with volumes in the range from 1 to 260 nl, the coefficient of variance (CV) is typically about 5%. As drop volume increases, however, variation becomes less of a problem; for drop volumes above 10 µl, CVs are commonly less than 1%.
Note that the valve-open time must be adjusted to accommodate different drop volumes. For example, it takes longer to form a drop of 1.0 µl than it does to form one of 10 nl. For a drop of 1.0 µl the required valve-open time is approximately 2 milliseconds, a relatively slow cycle that limits the operating frequency of the stepper motor to a maximum of about 500 Hz. Higher operating frequencies can be used when the system is dispensing smaller drops that require a shorter valve-open time. Since stepper motors or servomotors are capable of being operated at relatively high frequencies, over 5000 Hz, valve-open time can be programmed to deliver drops over a wide range of volumes. By combining programmable drop volumes and the use of high-frequency bursts of drops, users can obtain a wide variety of dispensing volumes and patterns.
Line Dispensing. In the drop mode of operation, a volume of reagent is delivered at the end of a motion sequence. In the line mode of operation, reagent is delivered between motion end points. Operation of the line mode is based on programming into the system the desired volume per unit of length, the rate at which the target substrate is moving, and the valve frequency. The resulting drops can be physically separated or overlapping, depending on the ratio of the valve frequency to the rate of motion. This relationship is described by the following equation:
Drops/cm = Valve frequency (Hz) / Rate of motion (cm/sec)
Since line volume is a programmable parameter, the volume of individual drops in the line can be determined by using the following equation:
Drop volume = (Line volume/cm) / (Drops/cm)
The system can be operated in a synchronous or open-loop mode. In the synchronous mode the system dispenses a drop volume equal to a harmonic of the step volume (syringe volume/syringe resolution). In open-loop mode, the user can arbitrarily select a drop volume to be dispensed, and thus produce drops that are nonharmonic.
Figure 3. In a positive-displacement dispensing system composed of a syringe and ink-jet valve, a volume of fluid can be delivered either as a single drop (a) or as a burst of smaller drops (b).
Different drop volumes and drop densities can be combined to achieve the same linear concentration (see Table I). By the same token, the pattern dispensed by the system can be varied by altering the drop density. This is a useful feature for diagnostic applications, where the format of many tests requires the dispensing of antibody or antigen lines onto a membrane. Figure 4 shows five lines dispensed using a 20.8-nl drop, but varying from well-separated drops at a drop density of 0.4 µl/cm to a well-defined continuous line at a drop density of 1.2 µl/cm. The sharpness of the drops is a manifestation of the very rapid binding of the protein to the target membrane, which occurs essentially on impact.
|Drop Volume (nl)||Drop Density (no./cm)|
Table I. When dispensing a line of reagent onto a membrane, linear volume is a function of both drop size and density. Each of the drop and density combinations shown here will result in dispensing a line of reagent with a linear volume of 1.0 µl/cm.
Physical Chemistry Considerations. Although the Biojet system is not as sensitive as conventional technologies to the physical chemistry of the reagent, those properties still affect dispensing by limiting drop formation and causing flow resistance in the fluid lines. The greater the viscosity of the reagent, the larger the minimum drop size achievable by the system. Similarly, with increasing viscosity, valve-open time needs to be increased to accommodate the slower rate of fluid movement. Reagents with viscosity up to about 50 cp have been successfully dispensed with a lower limit on drop sizes of 10.4 nl.
Although the system can dispense such heterogeneous solutions as gold and latex colloids, glass particles, cells, bacteria, and so on, there is a practical limit to the size of particles that it can handle. Reagents that include particles greater than 100 µm in size can begin to clog the system's fluid path, and are therefore not suitable for dispensing by this system.
Aspiration and Dispensing
In many applications, the reagent to be dispensed is available only in small volumes, and it is desirable to aspirate it from a holding format such as a microwell plate for subsequent dispensing. This can be accomplished by using the syringe as a pump and immersing the dispenser nozzle into the microwell with the valve open. In this case a hydraulic fluid is used to actuate the pump, and can also serve as a wash fluid between aspiration steps.
Figure 4. Line dispensing of a bound protein onto a nitrocellulose membrane using a set drop size of 20.8 nl, but with different drop densities: (a) 0.4 µl/cm; (b) 0.6 µl/cm; (c) 0.8 µl/cm; (d) 1.0 µl/cm; and (e) 1.2 µl/cm.
When using the Biojet system for aspiration and dispensing, minimum drop size is affected by the length of the nozzle needed to aspirate the reagent. The longer the nozzle, the greater the minimum drop size achievable by the system. Data for the dispensing of an aqueous reagent with one valve configuration show that a nozzle length of 10 mm will limit minimum drop size to 4.16 nl, while a nozzle length of 36 mm will limit minimum drop size to 10 nl (see Figure 5). Flow resistance also increases with increasing nozzle length.
Another consideration during aspiration and dispensing operations is the possibility that the reagent can be diluted by hydraulic fluid. This problem can arise when drag along the inner walls of the fluid path causes hydraulic fluid to linger in the orifice, where it mixes and diffuses during aspiration, and is subsequently dissolved into the reagent. The dilution effect is very sensitive to aspiration parameters.
During aspiration and dispensing operations, the volume being handled can affect the accuracy of dispensing, resulting in the wastage of small amounts of reagent (see Table II). This occurs because fluids in nanoliter and picoliter quantities have a much larger ratio of surface-area to volume than do fluids in microliter quantities. As a percentage of the total volume being handled, however, the amount of potential wastage is still very small and quite acceptable for most applications.
Table II. Accuracy of aspiration and dispensing operations for volumes in the nanoliter range. After aspiration of the indicated volume, a single drop was dispensed into a microwell with 200 µl of water. The reagent aspirated was a yellow food dye, and absorbance was measured using a plate reader at a wavelength of 450 nm.
Diagnostics. Typical diagnostics applications for this system are the dispensing of line patterns for immunoassays, and dot patterns for flow-through devices and biosensors. The system can be adapted to web operation by using a tandem syringe pump configuration that provides continuous dispensing. One of the important trends in diagnostics is the move toward quantitative test formats, which require the dispensing of precise amounts of reagent. The ability of this system to execute programmed dispensing of precise drop volumes will be very useful for the manufacturing of such tests.
Bioarrays. A very active area of research and development involves the use of high-density arrays of different reagents. Arrays of this type are being used for screening applications in a variety of fields, including diagnostics, drug discovery, and genomics.
Creation of these arrays requires aspiration and dispensing of hundreds or thousands of compounds, with drop sizes ranging from 100 pl to 50 nl and center-to-center placement as close as 50 µm. Often the reagents used for such arrays are available only in small volumes on the order of 5 to 20 µl. This system can be provided in linear arrays of eight channels on 9-mm centers, suitable for aspiration from 96- and 384-microwell plates. At present, the Biojet technology can dispense drop volumes down to 4.16 nl.
Figure 5. Minimum drop volume of an aqueous-based solution as a function of nozzle length, using a solenoid valve as the dispensing element.
High-Throughput Screening. To reduce costs and increase the throughput of compound screening for new drug discovery, many pharmaceutical firms have expressed interest in reducing assay size and increasing microwell plate density. Microwell plate densities up to 9600 with assay volumes down to 200 nl have been demonstrated (see Figure 6).4 The wells of a 9600-well microplate are tapered, with a top dimension of 1.0 x 1.0 mm and a bottom of 0.5 x 0.5 mm. To fill these wells with reagents, the line mode of operation is used with drop density set at 1—4 drops/mm and rate of motion set at 50 mm/sec. Using these parameters enables the tapered shape of each well to capture a consistent number of drops, thus filling each well with the same precise volume of reagent. The example shown has a measured CV of less than 10% for all wells. Using four independent Biojets in line mode, the entire 9600-well microplate can be filled in about one minute.
Figure 6. A protease assay in 9600-well format, typical of new approaches to high-throughput screening techniques. Using a line-scan process, all wells of this plate can be filled with 200 nl of reagent in about one minute.
Other pertinent applications of the technology include assay development for the emerging 1536-well format and for simplification of compound serial dilutions. The latter is made possible by the dispensing system's large dynamic range, which enables it to deliver a 1000-fold range of volumes.
Advances in IVD technologies, including the rapid growth in the field of miniaturized point-of-care tests, are continuing to challenge the capabilities of existing processing systems. IVD manufacturers have expressed a clear need for dispensing equipment that can operate at very high speeds while offering the capability of in-process inspection. They also require equipment that can produce small drop volumes in the picoliter range, with an ability to handle highly aggressive solvents such as tetrahydrofuran (THF).
Dispensing methods are keeping pace with current trends in the field of diagnostics through the development of advanced systems capable of processing an increasing range of reagents. Typical of recent successes in the field are improved piezoelectric dispensing, pin transfer technologies, and the combination of positive-displacement pumps with solenoid-valve dispensing elements as described in this article.
Still greater research will be necessary to improve processing consistency, increase throughput, and cover an even greater range of desirable reagents. Key improvements that may soon be seen include greater processing speed, two-dimensional dispensing, higher resolution to produce smaller drop sizes, and software that will enable systems to dispense more-complex patterns.
1. Tisone TC, "Quantitative Aspiration and Dispense in the Nanoliter Range for Drug Discovery Applications," presented at the IBC Conference on Microfabrication and Microfluidic Technologies, San Diego, August 7—8, 1997.
2. Lemmo A, Fisher J, Geysen H, et al., "Characterization of an Inkjet Chemical Microdispenser for Combinatorial Library Synthesis," Anal Chem, 69:543—551, 1997.
3 Papen R, "Nanoliter Dispensing for Drug Discovery," presented at the IBC Conference on Microfabrication and Microfluidic Technologies, San Diego, August 7—8, 1997.
4. Oldenburg K, "Plate Design, Image Analysis, and Liquid Handling for Miniaturization and Ultrahighthroughput Screening of Combinatorial Chemistry Libraries," presented at the IBC Conference on Drug Discovery Technology '97, San Diego, August 11—14, 1997.
Thomas C. Tisone is vice president for R&D and engineering at Bio-Dot, Inc. (Irvine, CA). Photo by Roni Ramos Photo courtesy of K. Oldenburg, Dupont Merck