Direct Sampling for Microwell Plates
Smart Ways of Biological Liquid Processing
- Fig. 1: Function principle of the i-doT
- Fig. 2: Droplet at the bottom of a well and sample arrays on glass
- Fig. 3: Minimum pressure pulse duration for different viscosities
- Fig. 4: Cell proliferation
- Fig. 5: Testplate with molded inserts
- Dipl. Ing. Andreas Traube, team leader production and process automation, Fraunhofer IPA
- Dipl- Ing. (FH) Tobias Brode, specialist for micro dosing systems and miniaturized liquid handling, Fraunhofer IPA
- Dipl.-Biol. (t.o.) Christian Reis, specialist for bio-adaptive automation systems, Fraunhofer IPA
One of the basic steps in the field of bioprocess engineering is the handling of liquids. The processing of those mediums as well as the cleaning of the dosing system is an important time, cost and quality factor for conventional systems. The Fraunhofer Institute for Manufacturing Engineering and Automation (IPA) developed a method that avoids this extensive process. This method allows a fast, cost-effective and flexible accomplishment due to a compact device, which performs the majority of the pipetting steps fully automated.
Due to an increasing number of assays and to reduce time and money, many laboratories start to be more and more interested in high grade automation. At the same time a high flexibility is needed for the automated processes in the laboratories. Therefore, in the future procedures and methods will be needed that cover a broad range of applications for as many different variants as possible .
The developments in the field of modern biotechnology tend to use a minimum volume for assays, while simultaneously increasing the assay diversity. E.g. protein and nucleic acid assays are used for highly parallel testing . Microarrays are clusters of very small drops that are dispensed on a suitable carrier in strictly predefined intervals. The "dispersion" is carried out either tangentially due to the adhesive force on the drop surface, or as a non-contact dispersion. During this so-called "non-contact spotting", the substance drops, which to be dosed are generated on the die opening of the chosen dispenser by very small pressure changes and with adjustable speed and size . For piezo-based spotting procedures, sample volumes in the picoliter range are used to create the sensor surfaces of the biochips. In other branches of biotechnology a trend towards the minimization of volume is noticeable as well. Due to high costs, the tendency to minimize volumes while simultaneously increasing the number of arrays is getting stronger, especially for antibody or cell-based assays. For the active ingredient screening in pharmaceutical drug development, still volumes in the upper nanoliter range are used.
Automated aids for liquid handling are already used in many laboratories today.
With an increasing grade of automation the devices range from manually operated electro-pneumatic pipettes to investment intense, interlinked robotic constructions for high-throughput use or bio productions. Cross-contaminations between different liquids are avoided in the pipetting system by using disposable pipette tips. If reusable metal pipettes are used, the pipette needles are cleaned in additional time consuming process steps .
For micro dispensing pipettes in the pico- to nanoliter range, the geometries of the cavities change inevitably due to the necessary integration of an electro mechanic converter for drop generation. For drop generation the wetted sections of the micro dispensers are optimized in a micro fluidic way. The small resulting wetted sections as well as the integrated actuating elements cause a difficulty when it comes to cleaning and rinsing. The creation of a disposable system for micro dispensers is impossible. Therefore, the process steps to avoid cross-contamination have to be realized through automated devices, which make them expensive.
Since the processing of liquids is often carried out in micro well plates, and those therefore serve as basic format for further processing, this format was chosen as basis of the "immediate drop on demand technology (i-doT)".
Immediate Drop on Demand Technology (i-doT)
The Fraunhofer IPA developed a method reducing or even eliminating many disadvantages of the conventional automated systems. From current micro well plate formats (96, 384 or 1536), the liquids can be spotted directly and cross-contamination free onto assay carriers or into other reaction tubes in exactly adjustable amounts. For this, nozzles with a diameter of 50 µm-125 µm are installed. This diameter is chosen to keep the liquid to be dispensed from leaking from the well under normal pressure, when the capillary pressure builds up in the nozzle. For a contact angle of water on regular plastics between 82° and 65° (with a bore diameter of 50°) filling levels of 41-124 mm are resulting. Since in standard micro well plates the typical well height is approx. 11 mm, the plate stays proof for the maximum filling level and materials like polypropylene and polystyrene for liquids with a surface tension of approx. 72x10-3 N/m.
The procedure for releasing the defined assay amount through the nozzles at the bottom of the well is based on a short compressed air pulse with a pulse length of 0.5-5 ms (fig. 1). This considerably exceeds the capillary pressure in the nozzle. With an optimized adjustment of the pressure impulse duration and the pressure, drops with a defined volume (2-10 nL) and repeatable accuracy can be generated. For testing the procedure, quick-release valves were used to generate the pressure impulse, which allow cycle times under a millisecond. For the generation of pressure, compressed air was used. To be able to approach individual wells of the microwell plate, a sealing punch is attached which is traversable in y-x-z direction. It approaches the individual wells, attaches as a seal and thus provides a link between the quick-release valve and the well. Below the microwell plate another x-y-z traversing unit is installed, which moves suitable object slides into the designated position underneath the microwell plate. This way, every generated drop can be disposed at any arbitrary spot on the object slide. Due to the high-precision positioning of the traversing unit and the merely slight angular misalignment of the drops, spots with a diameter of 150 µm can be generated on object slides with a position accuracy of 10 µm.
The smallest rugged drops that could be generated by this procedure have a diameter of approx. 130 µm. This complies with a volume of 1.2 nl. Therefore, up to 20,000 spots can be generated on a glass slide with a size of 25 mm X 75 mm which complies with a spot distance of approx. 300 µm (fig. 2). Liquids with a viscosity between 0.8 mPas and 1,000 mPas can be processed with this procedure (fig. 3). For liquids with a density of approx. 1x103 kg/m3, the surface tension should not be under 20x10-3 N/m, otherwise the capillary pressure of the cavity is not sufficient to seal a well.
The big advantage of the developed system is the high flexibility and speed of the procedure. Due to the high costs of every dispenser system, the parallelization of piezo dispensers is economically limited.
With the built in kinematics up to 12,000 spots can be produced every hour. Compared to conventional systems with the changing of pipettes or the purifying process, this system is highly time saving. If more than one punch is used or if several wells are approached through one well at the same time, the processing speed can be increased even further.
After printing HELA cells with this system, very few dead cells were visible. Only a few hours after, a significant proliferation was observed. The printing process doesn't influence the viability of the cells (fig. 4).
Since every i-doT well possesses its own nozzle and thus cross-contaminations between the individual mediums are impossible, depending on the plate format up to 1.536 different liquids can be printed without changing the nozzle or purifying. The used plates with the nozzles can be produced as disposable products through injection molding (fig. 5).
Other important procedures of this technology are available for laboratory automation. With pressure pulse frequencies up to 400Hz, rugged drops can be generated. Thus it is possible to extract a larger amount of liquids from the well and release it into a different assay container. With the new procedure it could be possible to position another micro well plate of the same or an arbitrary different format instead of the object slide, and thus to create either a clone of the original micro well plate or e.g. dilution series or mixtures in every desired combination.
Accordingly, this technology provides an alternative for the creation of micro arrays with densities up to approx. Three spots per mm, or for the reformatting between different microwell plates.
 Traube A. and Brode T.: Prozesstechnik & Automation, Sonderausgabe Select März, 26-29 (2008)
 Müller H.J. and Röder T.: Der Experimentator - Microarrays, 1.Aufl., München: Elsevier GmbH 2004, 15 ff.
 Rensch C.: Creation of Small Microdrops, MicroFab Technologies Inc., October 2006
 Chmiel H.: Bioprozesstechnik, 2. Aufl., München: Elsevier GmbH 2006, 213 ff.