Creating and Dispensing Small Liquid Volumes

tte controlled dispensing of very small liquid volumes was first demonstrated by Elmqvist (in the context of printing) in the Siemens-Elema Minograf recording mechanism (US patent 2,566,443, issued September 1951). Important factors in the dispensing of small liquids volumes are:

- Dynamic range of the dispensed volume

- Dispensing frequency (determines throughput)

- Precision and accuracy

- Linearity

- Reliability

- Ease of operation and maintenance

- General compatibility of surfaces and liquids(compatible with labile compounds)

tte preferred size range for (protein) droplets is between 20 pL and 20 nL, as the total trial volume should be low to a realize significant advantage over classical methods, tte manual, classical, dispensing of small volumes is by pipetting, but below a volume of roughly 200 nL pipetting becomes notably inaccurate and unreliable. Although manual dispensing can be used for small volumes, convenience and accuracy rules out their use in high-throughput experimentation. Low-volume manual dispensing in protein crystallization was reported by Yeh for drops above 100 nL using a handheld nanoject pipettor with an error of the order 5-9%. For drops smaller than 100-nL volume the error rises rapidly (Yeh 2003). For most applications a standard error of 5% is considered the upper limit (Rose 1999). As manual dispensing is neither accurate nor convenient at volumes below 100 nL, especially when variation in droplet composition is essential for the experiment, different methods are clearly needed, ttree established methods used in the field that can dispense in the nanoliter and picoliter ranges are the inkjet, electrospray and pintransfer methods.

Inkjet Technology

Several dispensing systems in protein nanocrystallization have been described in the literature (Stevens 2000; Bodenstaff et al. 2002; Howard and Cachau 2002; Krupka et al. 2002; Kuil et al. 2002; Santesson et al. 2003; Blundell and Patel 2004). Inkjet nanodispensing involves application of a force - electrical, thermal or acoustic - that generates a pressure wave through the fluid, tte liquid stream created is allowed to escape through a small orifice. When the liquid passes through the ori-

Acoustic Dispensing

Fig. 1.3. Piezoelectric dispensing versus electrospray dispensing. In the piezoelectric dispensing method {left) a pressure wave is generated that allows detachment and propelling of the droplet from the fluid body. Electrospray dispensing uses a different principle: an electric potential difference pulls droplets out of a fluid body. (From Bodenstaff et al. 2002)

Fig. 1.3. Piezoelectric dispensing versus electrospray dispensing. In the piezoelectric dispensing method {left) a pressure wave is generated that allows detachment and propelling of the droplet from the fluid body. Electrospray dispensing uses a different principle: an electric potential difference pulls droplets out of a fluid body. (From Bodenstaff et al. 2002)

fice the pressure difference allows the stream to overcome the surface tension forces and to be ejected as a drop. In a continuous inkjet the liquid supply is pressurized sufficiently to create a jet. ^e breakup of the jet can be synchronized by applying a periodic modulation of the velocity of the fluid exiting the nozzle. One of the most successful applications of the continuous inkjet technology is the Hertz continuous mist inkjet. In this method charged drops with volume of about 3 pL are produced at very high drop repetition frequency. Although this method is very fast and allows for high throughput, it has not been applied for protein crystallization. In drop-on-demand inkjet methods the forces to overcome surface tension forces and emit a drop or a cluster of drops are generated in response to a signal, ^e liquid supply is not sufficiently pressurized to form a (continuous) jet. ^e liquid is held in a nozzle, forming a meniscus, and remains in place until some force overcomes the inherent surface tension that keeps the liquid together, ^e commonest approach is to suddenly raise the pressure on the liquid, propelling it from the nozzle to the surface. It is also possible to pull the liquid out of a nozzle by an attractive force overcoming the surface tension (Pond 2000). For protein nanocrystallization the precise positioning and timing of the droplet deposition is of great importance; therefore, the drop-on-demand method of dispensing appears more suitable than the Hertz technologies. In view of the required drop volume, the precise positioning and the nature of the dispensed liquid, the relevant technologies are piezoelectric, electrostatic and acoustic drop-on-demand dispensing. To dispense solutions containing proteins that are possibly heat-sensitive, the bubble jet technology is considered less suitable. In piezoelectric dispensing a piezocrystal changes its shape in response to an electrical pulse, ^is results in "squeezing" a glass capillary and thus creates a pressure difference, either by opening a valve leading out of the pressurized container or by pushing against the fluid. As a result a drop is created "on demand." ^is type of piezoelectric dispensing is known as "squeeze mode." Various other methods such as the "bend", "shear" and "push" modes have been developed and differ in aspects less relevant for our purpose (Pond 2000). On-demand piezoelectric dispensers can typically create single drops in the picoliter size range and have been successfully used to dispense liquids and solutions with various properties in volumes as small as 0.3 pL (Howard and Cachau 2002). With present-day technology it is possible to very reliably produce droplets with a volume of 25 pL or more, ttese can be produced at a rate of at least 1,000 droplets per second, tte range of viscosities that can be dispensed with piezoelectric inkjet technology ranges from 0.4 to 100 mPa s, e.g., 100 times the viscosity of water at 20°C. Ms somewhat limited viscosity range is extended by the use of electrospray dispensing, where an electric potential difference is used to pull droplets out of a fluid body (for a review see Rohner et al. 2004). tte viscosity of the fluids and the presence of detergents are less relevant for electrospray dispensing compared with piezoelectric dispensing. However, there are currently no commercial dispensing stations that make use of drop-on-demand electrospray methods although electrospray dispensing has significant advantages for the dispensing of very viscous liquids, tte use of pulsed electrospray is relatively new (Wei et al. 2002), and recently we constructed several prototypes that can successfully dispense viscous liquids on flat substrates. A schematic comparison between the setup for piezoelectric dispensing and electrospray dispensing is given in Fig. 1.3.

A major problem in the reliable operation of any dispensing system is the problem of clogging, e.g., accumulation of solid material, given the small size of the orifice and the nature of the components to be dispensed. Clogging of the orifice starts and often results in drop misdirection with a disastrous effect on the overall reliability. Careful design of the chamber and orifice, including the proper choice of materials (and their wetting properties), results in reliable operation. For prolonged reliable operation good cleaning procedures and the purity and stability of the liquids to be dispensed are crucial.

Acoustic Dispensing

A novel technology that avoids the clogging problem is acoustic drop-on-demand dispensing where the free surface of a liquid is disrupted by a strong acoustic field. If the acoustic energy is well focused into a small volume of liquid near the surface, a drop of variable size can be ejected, tte droplet size depends on the acoustic energy field, the acoustic frequency in combination with the liquid properties. In acoustic droplet ejection (ADE), nanoliter or picoliter droplets are ejected from a conventional microplate by means of the acoustic energy generated by a piezoelectric transducer. tte energy is focused via acoustic lenses on the surface of the liquid, causing a droplet of precise volume to be ejected without any physical contact between the acoustic device and the liquid being dispensed. Drops are collected on another surface (e.g., a microplate) positioned in the path of the droplet, tte "nozzleless" ADE method avoids the reliability issues associated with the small orifice such as clogging followed by drop misdirection. As acoustic ejection is a noncontact method, cross-contamination between samples caused by the transfer device - a common problem in liquid handling - is largely avoided. A smallest drop volume of 2 pL is possible and the volume can continuously be adjusted up to 40 pL. tte acoustic drop-on-de mand dispensing technology can generate variable drop volumes at megahertz rates. ADE can dispense a wide variety of solvents, DNA, proteins and even live mammalian cells without detectable loss of activity or viability (Ellson 2003). Although acoustic dispensing has not been applied so far in protein nanocrystallization, the high speed and contactless mode of operation are substantial advantages, tte major advantage of ADE is the fast and powerful transfer of liquids. It is less clear how suitable the method is when the total amount of material is very limited and has to be dispensed in nanoliter volumes as in protein crystallography.

Fast Solenoid Valve Technology tte fast solenoid valve microdispensing method couples the accuracy of a stepper-motor-driven syringe pump with the high-speed actuation of a microsolenoid valve, tte syringe creates a steady hydraulic pressure within the system fluid, essential to obtain consistent and accurate droplet sizes. For a given pressure the desired

Simple Hydraulic System Syringes

Fig. 1.4. Fast solenoid-syringe method, a In the aspirate dispensing mode liquid is collected from microvials and redistributed into nanovials. This mode is used for small volume rearrangements. b In the bulk dispensing mode a larger volume is aspirated and distributed into the microvials. This step is used to rearrange moderate volumes. As shown in Fig. 1.7 the Cartesian instrument shows good linearity in both dispensing modes. (From Rose 1999)

Fig. 1.4. Fast solenoid-syringe method, a In the aspirate dispensing mode liquid is collected from microvials and redistributed into nanovials. This mode is used for small volume rearrangements. b In the bulk dispensing mode a larger volume is aspirated and distributed into the microvials. This step is used to rearrange moderate volumes. As shown in Fig. 1.7 the Cartesian instrument shows good linearity in both dispensing modes. (From Rose 1999)

drop volume is obtained by choosing an appropriate valve opening time, tte technology can be used in two different ways, aspirate dispensing and bulk dispensing. Using the syringe pump, either the sample can be aspirated from a source and then deposited into a destination, or the sample can be introduced through the entire fluidic path and dispensed in a continuous fashion. A schematic view of the bulk and aspirate fast solenoid dispensing mechanisms is given in Fig. 1.4 (Rose 1999). tte system is robust and adequate for setting up crystallization in the nanoliter range. In this method the potential of cross-contamination of the dispensed liquids makes a washing step between dispensing different liquids essential, tte use of parallel dispensing nozzles each dedicated to a single fluid reduces such demands. As in most dispensing technologies, calibration of the correct parameters in the dispensing process is needed for each solution.

Pin-Transfer Technology

Pin-transfer technology transfers liquid from a source to a location using a solid pin. tte process requires dipping a pin into a sample and taking it out. A small volume of liquid remains on the tip of the pin and by placing the pin on a solid surface the liquid is dispensed, tte major advantage of this technology is that the pins are simple and relatively inexpensive, while volumes in the low-nanoliter range can be transferred, tte pin method has limited flexibility as the pin dispenses a fixed small volume that depends on the tip properties and geometry in combination with the liquid properties, ttis limits the use in applications requiring variable volumes. Another disadvantage of this technology is that clogging can occur, especially when suspended particles are relatively large with respect to the tip or the gap in the pin. If throughput and cost are more important parameters than high precision and accuracy, pin-transfer technology is a possible alternative to noncontact dispensing. When a screen using fixed volumes of liquids optimized for protein crystallization has been identified, a very fast system with disposable pins could be mass-produced.

Comparison of Liquid Dispensing Methods

Important aspects of the dispensing techniques related to protein crystallization are summarized in Table 1.1. Some highly relevant factors for protein crystallization cannot be compared easily for all techniques and all strategies. As an example, the dead volume of a dispensing system is not a problem for most liquids but for the component of most interest, the protein to be studied, it is highly relevant. No matter how accurate and reliable a dispensing system is, if it is necessary to inject some hundred microliters of precious protein the system it will be of little to no value. Evaluation and reduction of the dead volume is an issue that is not widely addressed in the field of nanodispensing, which mostly focuses on throughput and accuracy. If one would like to compare the amount of protein needed per trial between methods, the dead volume should be included. Equally relevant for all dispensing techniques

Table 1.1. Comparison of nanodispensing techniques. Some important aspects of different dispensing techniques related to protein crystallization are compared. Electrospray has a great potential in protein crystallization, but it is not yet commercially available

Property

Pin method

Piezoelectric inkjet

Thermal inkjet

Acoustic inkjet

Electrospray

Fast solenoid valve column

Driving force

Mechanical

Pressure

Heat

Sound wave pressure

Electric

Pressure (release)

Biocompatible

Yes

Yes

No

Yes

Yes

Yes

Viscous solutions

No

No

Yes

No

Yes

No

Accurate, reproducible

No

Yes

Yes

Yes

Yes

Yes

Small volumes (nanoliterand subnanoliter)

Yes

Yes

Yes

Yes

Yes

No

Low energy transfer to sample

Yes

Yes

No

Yes

Yes

Yes

is the (cross-) contamination of the dispensing system that ultimately results in unreliable operation and failure, tte problem of tip contamination has been addressed in the Mosquito dispensing system where an automated system simply discards the tips after use for a single compound. An elegant and rapid way of cleaning and drying for nondisposable tips and equipment is still to be found. In this respect the ADE method seems a viable method as it eliminates the need for disposing and/or washing steps altogether.

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