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Droplet by droplet – nanodosing of the highest quality

Researchers at the Fraunhofer IPA in Stuttgart have developed a new method for the automated dosing of liquids in the micro- and nanoscale. The “i-doT” system avoids the use of pipette tips, is suited for high-throughput applications and allows the contact-free spotting of samples, thereby avoiding contamination. The sample droplets are transferred into microtitre plate well boreholes through the application of precisely dosable pressure pulses.

These microarrays were “printed” with the i-doT system. © Fraunhofer IPA

Rising costs have led to the use of microtitre plates with higher numbers of wells per plate: after the standard 96-well plates, 384-well and subsequently 1,536-well plates have entered the market. This has led to a reduction in sample volume, although more than five millilitres are still needed for the 1,536-well plates. Since the high-throughput methods used in biotechnology are associated with exorbitant costs when large volumes need to be handled, new nanodosing methods are in great demand. Besides the accurate dosage of the samples, it is important that the samples are transferred under sterile conditions, which considerably reduces the risk of contamination.

With these requirements in mind, Tobias Brode and Andreas Traube from the Fraunhofer Institute for Manufacturing Engineering and Automation IPA initiated the development of i-doT (immediate drop on demand technology). The two engineers - Brode studied microsystems engineering in Berlin and Traube mechanical engineering in Stuttgart - thoroughly familiarised themselves with biotechnological methods, in which their excellent contact with the biotech specialists at the Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, which is also located in Stuttgart, was of great help. "We see ourselves as a kind of "bioneers", as mediators between biology and engineering with a huge dollop of pioneering spirit," said Brode.

”Bioneering“ facilitates laboratory work

The two researchers came up with the compelling idea of drilling tiny boreholes into the wells of microtitre plates. These boreholes are so small that the capillary forces acting in them seal the opening under the pressure exerted by the normal fluid level in the well. However, when a tiny air pressure pulse is exerted from above, the capillary forces temporarily change, thereby enabling a small droplet to leave the opening. Up to 400 droplets can thus be produced per second, confirmed the developers. Different droplet volumes can be achieved by altering the diameter of the opening. Openings with a diameter of 110 micrometres can create droplets of four to seven nanolitres, depending on the air pressure applied. The team of researchers have reduced the diameter to 70 micrometers, which allows them to produce droplets with a volume of as small as one nanolitre.

Functional principle of the contact-free and hence contamination-free i-doT dosing system. © Fraunhofer IPA

“The quality of the boreholes is decisive for obtaining reproducible droplet volumes. The use of stroboscopes helps us in assessing the homogeneity of the size of the droplets. The error rate of the dosing accuracy is below two per cent per well. Thus, the i-doT system is no worse than pipettes and the majority of researchers use higher volumes,” said Brode. Thus, i-doT is a real alternative to traditional pipetting robots and has clear advantages in terms of costs and handling: the researchers do not need to clean or exchange pipette tips, the i-doT plates are easy to produce and are hence inexpensive disposable products. The plates can be used for example in drug screening: “Up to 300,000 tests can be carried out per day when i-doT is used in high-throughput applications,” said Brode.

From plate to plate with a high throughput

A pressure pulse leads to the release of droplets. © Fraunhofer IPA

The new method is also suitable for microarrays. "We have been able to show that samples are not carried over when spot distances as small as 400 micrometres are used. Thus, millimetre-scale distances between the spots are even less of a problem and we can use i-doT to print arrays on slides or in microlitre plates." In problematic cases such as very viscous solutions or samples containing components of low solubility, i-doT has already been shown to be more robust than pipetting robots. "When protein solutions are used, for example, it occasionally happens that individual components precipitate, or the surface tension and viscosity of the solution changes. This is a smaller problem for i-doT than for pipetting systems," said Brode. Relating to the viscosity of the solution, i-doT can in theory lead to satellite droplets; however this risk can be reduced by minimising the diameter of the boreholes: Brode explains: "The smaller the diameter of the opening, the better the transfer of energy to the droplets. This means that the range in which the correct pressure needs to be selected becomes broader and it is a lot easier to handle the system."

i-doT also enables the transfer of cells. "We have already succeeded in creating a regular cell lawn with i-doT," confirmed Brode. No matter whether HeLa cells, fibroblasts or keratinocytes are used - i-doT has proven suitable for accurately dosing all tested cell lines, and the cells proliferated well after transfer. "We did not observe any adverse effects," said Brode. The combination of the i-doT system with a cell sorter and cell counter system appears to be an interesting issue and an interesting challenge. "We have considered attaching a microcamera and a laser close to the opening and modifying the boreholes for sorting processes. We do not envisage any problems as far as the control of the system is concerned, but we have yet to find out whether it also works on the single-cell level," said Brode going to add "there are really so many ideas we would like to pursue in the future."

Establishing i-doT as basic technology and then commercialising it

At present, the two researchers are working feverishly on the further development of i-doT, before at some stage commercialising the system by setting up a company. “At the moment, we are in the comfortable position of being able to continue our research at the Fraunhofer Institute in order to optimise the technology and the devices,” said Brode. On 9th February 2010, the new bioproduction laboratory BioPoLiS was opened on the IPA premises, providing the i-doT team with the facilities to continue their work and speed up their progress. The two researchers are sure that “BioPoLiS is the perfect environment to further develop i-doT. We are able to work under sterile conditions, have access to compressed air supply systems and sufficient space to test our ideas and identify potential weaknesses in the system.”

The team's current achievements have already led to some success: Brode and Traube participated in the 2009 Science2Start competition run by BioRegio STERN Management GmbH and were awarded second prize for i-doT. The competition has provided motivation and support for the researchers in their plans to use their ideas for setting up their own company. Brode and Traube have set up a concrete plan and are pursuing it vigorously at the same time as remaining enthusiastic about the excellent working conditions at the IPA. “We have already been contacted by industry and will present the first commercial applications in 2010. We hope to have everything in place by 2012 enabling us to start our business,” said Brode.

Further information:

Fraunhofer Institute for Manufacturing Engineering and Automation IPA
Dipl.-Ing. Tobias Brode
Dipl.-Ing. Andreas Traube
Nobelstraße 12
70569 Stuttgart
Tel.: +49 (0)711 970-1257

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/droplet-by-droplet-nanodosing-of-the-highest-quality