Medical Device Link.

Originally Published IVD Technology July 2003

Laboratory Instrumentation

Parallel output ports on the Picotap, an on-chip reaction-well interface. These ports house Picopins, capillary uptake devices that can extract sample from microchips.

The IVD industry is moving toward the increased use of microfluidics. This technology offers the benefits of low cost and high throughput, providing parallel sample and assay analysis via disposable chips that can be fabricated from polymer, glass or quartz, silicon, and other materials that support the movement and processing of biological samples and associated reagents.1–3 Microfluidic components enhance both economy and productivity in testing by enabling parallel processing to be implemented on a single small substrate. Cost savings also are engendered by minimized use of biological materials, limited consumption of expensive reagents, and the employment of inexpensive disposable components and materials.

Microfluidics technology encompasses devices such as DNA microarrays, immunoassay chips, microchannel-based separation modules, and lab-on-a-chip or micro total analysis system (µTAS) devices.4–7 It supports applications in rapid pathogen identification, marker identification for diagnostic and therapeutic analysis, single nucleotide polymorphism analysis, and many other areas of diagnostic and therapeutic interest. But high-density microfluidics technology will not reach its full potential for adoption into high-volume laboratory diagnostic environments until microfluidic devices can be integrated easily into supporting laboratory automation platforms.

Figure 1. A rendering of a Picosocket, showing device construction and drop injection (inset). Click to enlarge.

Such integration currently is difficult because the fluid volumes for which existing diagnostic and laboratory automation and dispensing systems are suited are generally larger than the volumes processed by very-high-density microfluidic devices (50 nl or less). Commercially available dispensing robots typically can dispense accurately only volumes of 50 nl or more per droplet or dispense cycle. Microfluidic devices, on the other hand, may be able to support fluid volumes even within the smaller picoliter range. Most robotic systems also do not have components small enough to effect mechanical insertion or extraction of samples on this scale. Any integration of high-density microfluidic devices with standard liquid-handling equipment must support low-volume fluid transfer between the diagnostic instrumentation (which may include sample and reagent storage modules as well as detection or postprocessing analysis stations) and the microfluidic chips. Such integration ideally would accommodate fluid volumes below 50 nl per dispense cycle, with minimal liquid dead volume remaining after the transfer operation.

Figure 2. Dye-filled microchannel with coated ports. The inset is a close-up of the meniscus. Click to enlarge.

This article presents a novel way of interfacing robotic automation systems with microfluidic chips that overcomes previous impediments and achieves desirable results. The technology can be applied in capillary electrophoresis (CE), protein and DNA separation, microarrays, on-line gradient spotting, dispensing, serial dilution, and mass spectrometry. The article discusses the core technology and reports experimental results obtained in such applications as array spotting, direct injection into a microchannel, and formation of a sample plug for CE separation.

New Chip Interface Technologies

A small number of technologies for low-volume fluid dispensing and transfer have been introduced recently. For example, Picoliter Inc. (Sunnyvale, CA) has developed an innovative system to eject picoliter and nanoliter liquid volumes by means of acoustic energy.8 Designed for compound library transfer and cell transfer between high-density multiwell plates in the drug discovery environment, this technology provides dispensing of extremely low sample volumes, but does not address the challenge of transferring them into or out of the microchannel formats found on some microfluidic chips with zero dead volume.

Figure 3. Experimental separation setup for testing the operation of Picosocket port devices. Injected bands were about 100 µm wide. Click to enlarge.

Caliper Technologies Corp. (Mountain View, CA) has developed a microfluidics platform trademarked as LabChip that includes a device for transferring fluid onto a microfluidic chip from a fluid reservoir by means of capillary action.9 Its technology is highly effective in introducing a small volume of fluid to a microfluidic channel. However, a large footprint on the microfluidic device limits the ability to optimize the density and throughput of the chip.

Teragenics Inc. (Watertown, MA) has developed a portfolio of technologies for efficient integration of robotic automation and dispensing equipment with microfluidic components.10 Picogenics technologies include a method for transferring sample from a robotic dispensing station onto a microchip column or microwell (Picosocket) and a method for extracting sample from a microchip column or microwell (including Picotap, Picopin, and Picojector technologies). They support integration of microfluidic chips with pre- and postprocessing operations and feature zero dead-volume losses at the point of transfer. The technologies provide a particularly efficient integration of microfluidic-based sample preparation and separation processes with laboratory automation equipment.

Picogenics technologies are well suited for high-throughput, low-sample-volume diagnostic applications. Their ability to input directly onto and extract from microfluidic channels picoliter or nanoliter fluid quantities has been demonstrated and is discussed in the next section of this article.

Low-Volume Liquid-Input Technology

Figure 4. The reproducibility of five separate injections at two different concentrations, with electropherogram at right. Click to enlarge.

The Picosocket interface can be used to transfer sample volumes onto a microfluidic channel or reaction chamber without direct physical contact. The device may be characterized as a 200- to 100-µm tapered hole, or port, in the side of a microfluidic channel that has a hydrophobic coating along the shaft (see Figure 1). The footprint of the input port approximately matches the diameter of the microchannel.

Small fluid droplets 30 to 100 µm in diameter and about 200 pl to 2 nl in volume can be injected directly into the channel through the port by means of a low-volume dispensing station. The hydrophobic coating of the port shaft prevents injected sample from aggregating along the shaft. A meniscus forms at the bottom of the port (the roof of the chamber) when the channel is filled (see Figure 2). The device has been designed such that the burst pressure of the interface at the microchannel is 1–2 kPa, which is sufficient to ensure that material traveling down the channel does not escape through the port under normal (low-pressure) operating conditions. Injected droplets from a robotic dispensing station are able to penetrate the meniscus and flow directly into the channel.

Figure 5. A 32-channel CE chip incorporating Picosocket technology.

Operation of the port was tested via an experimental setup shown in which microdroplets were directly injected in a CE column by way of the input port using a conventional piezo dispenser (see Figure 3). A voltage of 600 V/cm was applied across the channel, and separation of bands was observed through use of a mercury-arc light source, a fluorescent emission filter, and a photomultiplier tube. Experimenters made a ladder of fluorescein-labeled 5-mer peptides and injected varying sample amounts and peptide concentrations into the same column. Electropherograms of the five separate consecutive injections were then produced (see Figure 4).
Experimenters were able to inject very narrow (100-µm) bands onto the separation column, and obtained band separation spacing of 13 mm at applied voltage of 600 V/cm using a PBS (phosphate-buffered saline) buffer. The coefficient of variation on measuring ratios of band peaks was better than 5%. The concept of using these ports with separation columns has been the basis of the development of an extremely high-density CE chip (see Figure 5).

Figure 6. A rendering of a Picotap, showing device construction and liquid pickup by means of a slotted microfabricated pin (inset). Click to enlarge.

The 32-channel, glass-based electrophoresis chip shown in the figure is only 1 cm wide. Thirty-two separate ports (within the gold band) are used to inject sample into each of 32 columns. The input socket makes this high-density CE device possible, because no sample wells or injection crosses are necessary to support on-chip separation. The amount of sample required per separation is also extremely small—just 1 nl per channel—owing to the fact that dead-volume losses are avoided. In addition, because of zero dead volume between injections, no flushing between uses of each channel is necessary.

A significant advantage of the demonstrated socket over conventional microfluidic interfaces is direct injection of droplets into a microchannel without the dead-volume losses seen in capillary and other input mechanisms. Experiments have confirmed that the technology facilitates direct injection into microscale CE columns of extremely low fluid volumes in the picoliter and low nanoliter range. In addition, the entire input sample is used when a droplet is injected into a microchannel via the port. This can be very advantageous in applications where reagent costs are high or where sample volumes are small and sample loss is unacceptable, such as certain forensic, DNA testing, and proteomics applications.

Figure 7. Picopin parts on a silicon wafer, with insets showing geometry detail of a single pin. Click to enlarge.

The device also provides a high-throughput injection mechanism. Dead time between injections can be small, since no flushing of reservoirs is required between sample injections. The Picosocket is a true random-access input device that can support nonsequential input into random microscale columns without additional overhead costs. Standard robotic sample-handling equipment can perform direct injection into the input port in high-volume laboratory settings.

The small footprint of the port provides the means to create high-density, low-cost microfluidic chips. Because no additional mechanisms are needed to insert sample into the microfluidic channels, on-chip channels can be tightly spaced. The input port also requires no supporting on-chip “real estate” because no on-chip reservoirs or delivery channels are necessary. The device adds negligible cost to the microfluidic component; it can be fabricated for less than one dollar per port (estimated) in volume manufacturing.

This low-input technology needs to be modified before it can be used in applications where capillary or reaction chamber pressure is extremely high. Use of the unmodified component in certain high-pressure sequencing or chromatography applications might be precluded. The technology also requires modification for use in applications where fluid surface tension is too low, such as when certain low-surface-tension solvents are used for separations or analysis.

Most diagnostic applications that can be envisioned as based on an on-chip microfluidic channel can be supported by the technology. It is well suited to serve as an input mechanism for CE (DNA and protein separation or affinity binding); narrow-band (100-µm) column injection on chip for CE without electrokinetic switching; gradient production applications; and microwell input applications.

Low-Volume Liquid-Extraction Technology

Figure 8. Experimental results of using a microfabricated pin to perform withdrawal and consecutive spotting of sample from an on-chip output port. Click to enlarge.

The microchannel input mechanism described has been demonstrated to efficiently transfer sample onto a microfluidic channel. Mechanisms for efficiently extracting material from microchips have also been developed and tested to perform as intended.

Contact-Based Methodology. The Picotap is an on-chip column interface, or output port, based upon the same physical structure as the Picosocket. This output port can be used to extract small sample volumes (200 pl to 2 nl) from a microfluidic channel or reaction chamber by means of a newly developed, microfabricated slotted pin, the Picopin. It consists of a 200- to 100-µm tapered port in the side of a microfluidic channel that has a hydrophobic coating along the shaft, similar to the design of the low-volume input device (see Figure 6). Each Picotap occupies only 0.04 mm2 of the chip and can be designed to function on the end of any electrophoretic column, on the low-pressure end of any pressure-driven column, or as an output port for a reaction chamber. Fluid or sample material can be extracted directly from a microfluidic channel or reaction chamber by inserting the microfabricated pin into the output port as shown in the inset portion of Figure 6. The sample that has been withdrawn may then be spotted onto an array or microwell plate.

Figure 9. Artist’s rendering of a Picojector, showing device construction and drop ejection (inset). Click to enlarge.

The slotted pin is a precisely dimensioned capillary uptake device that can be used to withdraw a 200-pl to 2-nl sample from an on-chip output port (see Figure 7). It is a silicon batch-fabricated component with a hydrophilic coating on the interior surface (in the capillary pickup or uptake region) and a hydrophobic coating on the exterior. The coatings, along with the precise dimensional control of the capillary pickup region afforded by silicon microfabrication technology, optimize the coefficient of variation and volume control of small pickup and dispensing volumes. Microfabricated pins can be used to create spots on an array as small as 50 or 100 µm in diameter. They also can be batch fabricated in arrays matched to 96-, 384-, and 1536-well plates at a low cost per pin, making them highly suitable for integration into high-throughput diagnostic systems that use standard microtiter plates.

A variety of experiments were performed to validate the capability of the Picotap/Picopin system to extract precise sample volumes from a capillary channel and to dispense the samples onto an array (see Figure 8). Selected liquid removal from an output port using a batch-fabricated pin was investigated, as illustrated in the figure. The tip of the pin was inserted into the output port (upper sequence in the figure) and used to extract a precise 300 pl of liquid via capillary force. The pickup volume was determined by the size of the slot at the bottom of the pin. Pickup volumes from 400 pl to 2 nl have been reliably demonstrated using the microfabricated pins.

The geometry and coatings of the silicon-based pins prevent fluid from accruing in areas other than the slotted region of the pin. When the pin touches down on a glass slide (see Figure 8, lower left), the fluid in the capillary region of the pin is released. An array of 75-µm dye spots created on a glass slide using one of the pins is shown in the lower right corner of the figure. This type of spotting is suitable for intermediate storage of reagent or samples on a microarray slide as part of a high-throughput diagnostic station.

This technology is cost-effective for many high-throughput diagnostic applications. The passive Picotap component adds little cost to an existing microfluidic chip—probably less than one dollar per port in volume manufacturing. Microfabricated Picopins have a lower high-volume cost model than stainless-steel pins owing to their batch production. Precision silicon batch fabrication technologies give the pins better geometry control than existing stainless-steel pins, as well as more-precise dispensing capability.

The features of this technology suit high-volume diagnostic applications requiring precision volume control and an interface between a microfluidic chip and automated downstream diagnostic or analytical operations. Compatible downstream processes include sequencing applications, generation of nucleic acid and protein arrays, applications requiring precise serial dilution (such as electrochemical, fluorescent, or other binding assays), and matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) or other downstream analytical techniques. The capillary uptake pins can be mounted on a robotic system to provide a high-productivity diagnostic workstation interface for a microfluidic chip via the microfluidic output port.

Figure 9. Artist’s rendering of a Picojector, showing device construction and drop ejection (inset). Click to enlarge.

Like the Picosocket, the Picotap and Picopin technologies also must be modified for certain applications, particularly those in which capillary or reaction chamber pressure is extremely high or in which fluid surface tension is low. Since the interface is a contact-based methodology, it is not designed for applications requiring noncontact transfer of sample.

Noncontact Sample-Transfer Technology. Noncontact sample transfer is necessary in diagnostic tests where treated surfaces, such as optical-quality deposition surfaces, could be damaged by contact-based systems and in applications where postprocessing from the microfluidic chip requires an immediate input of airborne droplets, such as direct injection of droplets to a mass spectrometry system.

A Picogenics technology has been developed that can be used to provide a noncontact interface between a microfluidic chip and a downstream analytical or diagnostic station. The Picojector performs the extraction of small samples (volumes in the low nanoliter range) from a microfluidic channel or reaction chamber by means of airborne transfer. The device has a familiar design, consisting of a 200- to 100-µm tapered port in the side of a microfluidic channel that has a hydrophobic coating along the shaft (see Figures 9 and 10). A membrane located beneath the ejection structure is pulsed to eject drops out of the on-chip channel or reaction well. The ejection structure can make use of any off-chip pressure pulse actuator; a piezo actuator is shown in Figure 9.
In experiments to determine the capabilities of this device, its substructures were fabricated on microfluidic-chip-based column arrays, and drops were ejected directly from the columns. Drops 300 to 400 µm in size were demonstrated to be ejected directly from these on-chip columns at speeds greater than 10 Hz. Drops of about 10 nl in volume were ejected by pressure actuation. The droplets have shown good size uniformity (see the Figure 10 inset). The ejection system is able to support ejection of droplets 200 pl to 40 nl in size.

The noncontact interface can be used to perform selective ejection of on-chip bands from microfabricated separation columns into a mass spectrometer or other system that accepts droplets. The device alternatively can be designed to function on the end of any electrophoretic column or the low-pressure end of any pressure-driven column. Again, this on-chip component is passive and inexpensive, thus lending itself to high-volume diagnostic applications involving on-chip separation and mass spectrometry, noncontact array spotting, and other noncontact postprocessing procedures.

Conclusion

Technologies for injection and withdrawal of picoliter through nanoliter fluid quantities from chip-based microchannels have been shown by experimental results to be able to interface macroscale, high-throughput robotics and diagnostic equipment with high-throughput microfluidic devices.

A microfluidic-chip input system can inject sub-1-nl sample volumes and narrow bands directly into an on-chip separation column or reaction chamber. It allows higher-density separation devices than previously possible to be created. Applications could include a high-throughput capillary electrophoresis system entailing a cost of less than 10 cents per separation, as well as on-chip reagent titration systems and on-chip, high-throughput proteomic separation systems.

The viability of several device technologies for outputting small fluid volumes directly from on-chip separation columns or reaction chambers onto multiwell plates, microarrays, or downstream diagnostic systems has also been demonstrated. These technologies provide a mechanism for contact- and non-contact-based extraction of sample from a microfluidic chip. They are compatible with conventional robotics and high-throughput laboratory diagnostics and offer an economical, small-footprint, and passive-structure methodology for direct extraction of biological samples from microfluidic chips. Potential uses for these technologies include low-volume dispensing from microfluidic chips to nucleic acid and protein arrays, transfer of separated bands from microfluidic channels for diagnostic and sequencing applications, and transfer from chip-based sample preparation or analysis devices to MALDI, mass spectrometry, and other downstream laboratory diagnostic systems.

References

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9. SA Sundberg et al., U.S. Pat. 6,086,825 and U.S. Pat. 6,090,251.

10. S Bohm et al., “Chip-to-World Interfaces for High Throughput Lab-on-a-Chip Devices,” in Micro Total Analysis Systems 2002, Proceedings of the µTAS 2002 Symposium, ed. Y Baba, S Shoji, and A van den Berg (Dordrecht, Netherlands: Kluwer Academic Publishers, 2002), 61–63. 

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