Liquid Handling for Reliable Assay Miniaturization

Why then, has realization of this goal been so slow and difficult? There are many reasons that one can highlight such as archaic protocols, unsuitable labware, problematic compound stores, and detection limitations, but perhaps the most important reason can be found in the lack of reliable low volume liquid handling. Here we explain a novel and robust approach to a liquid handling architecture that has shown itself to be reliable and effective for the tasks associated with assay miniaturization.

Limitations of Current Approaches

Traditional approaches to low-volume liquid handling technologies range from classical liquid handlers, employing syringe-based dispensing, to piezo-electric dispensers. Some offer a fixed volume at the expense of accuracy and precision while others promote a variable volume range at the expense of delivery or dead volume. An additional class of dispense-only devices use a flow-through approach. In these systems, reagent flows through the entire fluid path propelled by a backpressure source such as a pressurized reservoir, syringe drive or perlistaltic pump. In all of these flow-through systems, the entire system must be primed with the reagent and most utilize a dispense actuator in the flow path.

The limiting factor of traditional liquid handling techniques is the fact that they rely on low energy displacement. To perform a reproducible and accurate dispense, the last task of any pipetting action relies on a touch off. Classical displacement techniques do not have enough energy to break the surface tension of the last droplet. So a dragging action -- touch off -- is employed, either against the solid surface of a vessel or a liquid surface. Consequently, this technique is variable -- it varies with liquid properties, temperature, humidity, surface adhesion, and other factors. At larger volumes the variation is small enough to have little impact on the end result. However, when the total volume pipetted is small, nanoliters to single digit microliters, this variation has a significant negative contribution towards imprecision.

Advantages of Non-Contact Dispensing

Non-contact liquid handling relies on the combined use of rapidly actuated solenoid dispense valves, a controlled pressurized liquid source, and flow path control via hybrid valves. The speed and energy of the fluid displacement enables the surface tension of the liquid to break as it leaves the orifice, eliminating the need for a touch-off. The lack of a touch-off requirement in the dispense step eliminates variability. High-speed photography demonstrates the clean and exact start and stop of the fluid in a “ink-jet” solenoid-based dispenser.

High Speed Photography of 100 nL Dispense (1000 frames/second)

The following bar graph provides a typical example of the precision achieved with Innovadyne dispensing instruments at 100 nL:

Overall %CV Across Four Plates (100 nL Dispense, 384-well Plate)

An important added benefit of non-contact dispensing is speed -- the non-contact technique allows the delivery nozzle to accurately deliver the droplet above the target well and rapidly move to the next well. With the non-contact technique dispensing becomes independent of the substrate, eliminating many of the reproducibility problems associated with motion control. Plate processing times fall dramatically. Using Innovadyne 8-channel dispensing instruments, it is possible to deliver to all wells of a 96 well plate in approximately 5 seconds, 384 wells in approximately 7 seconds, and 1536 wells in approximately 14 seconds.

Isolation of solenoid valves from the sample path

The performance of solenoid-based delivery techniques relies on the speed of opening and closing of the valve. To be effective the actuation must be both rapid and reproducible. Many samples and reagents are either adsorbent (such as proteins) or particulate (samples marginally soluble in DMSO) and the introduction of these materials to the complex internal path of a solenoid valve can lead to deposits, obstructions, and wearing of the seal materials. Notice the many “unswept” areas of the solenoid valve below.

Valve deterioration from deposits and wear is not only costly, requiring valve replacement, but can also be difficult to diagnose because the effect can be a gradual deterioration of performance or intermittent failure (that is, no dispense). It can be very confusing to try to sort out the variation in the day-to-day reproducibility of the overall method when one is unsure whether the hardware or the actual chemistry is to blame. Innovadyne’s valve-free flow path ensures rugged performance.
Industrial strength pipetting for HTS

The incredibly high duty cycles of HTS and µHTS facilities, often 250 or more 1536-well plates per day, will expose any weakness in a technique or instrument. The precision of solenoid-based systems has long been recognized, but widespread acceptance has been tempered by concern regarding reliability and maintenance issues. Innovadyne has addressed this concern with the introduction of a proprietary hybrid valve architecture that includes a valve-free flow path, isolating the sample aspiration path from the solenoid dispensing valves. The technology is based on an architecture developed under a collaborative research agreement with the Oak Ridge National Laboratory. The architecture can be seen below:

Reagent Aspiration, using the Syringe Path

To aspirate an air gap, the tips are exposed to the atmosphere (not descended into the reagent tray), allowing air to flow into the tips. The valves are switched into position, allowing flow from the tips to the syringes. The syringes are pulled down, creating a vacuum, and system fluid flows from above the reagent tray to the syringes. Air is aspirated through the tips and system fluid flows toward the syringes. Next, to aspirate reagent, the tips are descended into the reagent tray, then the syringes are pulled down further. Reagent flows from the reagent tray to the syringes, separated from system fluid by the air gap.
The pressure path, using a pressure reservoir filled with de-ionized system liquid held at system pressure, is used to dispense reagent. The pressure path is the flow path from the pressure reservoir, via the micro-solenoid valves, to the tips, as shown:

Reagent Dispense, using the Pressure Path

The pressure reservoir contains system liquid, maintained by a digital pressure regulator (DPR) at a specified system pressure. To dispense reagent, the hybrid valve switches the flow from the syringe path to the pressure path, and then the micro-solenoid valves are opened and closed the requisite number of times to permit the desired volume of system liquid to flow from the pressure reservoir toward the tips. Note that the reagent does not itself flow through the micro-solenoid valves; system liquid does. The motion of the system liquid towards the tips displaces the desired volume of reagent out through the tip, and reagent is dispensed into the plate.

By isolating the critical flow-regulating device, the solenoid, from the sample flow path, this simple design eliminates the risks present in earlier systems and takes a major step forward in terms of robustness. The solenoid valves are now only exposed to de-ionized water at a constant pressure, allowing them to operate efficiently and effectively, as they were originally designed. Rather than lasting for only months, solenoid valves can now be expected to perform for years. In addition to improving robustness, the system design ensures that the aspirated sample never contacts moving parts and only encounters a very simple flow path. The benefits for difficult samples are clear. A wide range of reagent viscosities together with beads, cells, and complex mixtures have been pipetted successfully.

Technology for today and the future

Many disciplines are now beginning to apply miniaturization strategies to lower costs and improve efficiency. Innovadyne Technologies now supplies users throughout the Life Sciences, including genomics (PCR and sequencing), proteomics (MALDI and protein crystallography), HTS (Assay development and screening), biosensors and diagnostics with reliable, low-volume liquid handling, providing solutions for today’s applications with extensibility for future applications. Innovadyne Technologies: Leading the way in high precision dispensing.