The identification and characterisation of individual DNA and protein molecules is gaining in importance. A method developed by Dr. Gerhard Baaken and Prof. Dr. Jan C. Behrends at the University of Freiburg has the potential to be used by scientists to analyse a large number of single molecules automatically using nanopores. The MECA (microelectrode cavity array) technology can be adapted to a broad range of different applications.
“We are aiming to be able to measure anything related to ions and salt solutions using membrane proteins to do so,” said Dr. Gerhard Baaken from the University of Freiburg, talking about what led to the establishment of Ionera. “The name Ionera is connected with the slogan “… wherever ions flow” and represents a new era of electrophysiology applied to molecular analyses,” said the engineer.
Baaken studied microsystems technology at the University of Freiburg and was awarded his PhD by the Department of Microsystems Technology (IMTEK) in 2008. His doctoral thesis, which he did under the supervision of Prof. Dr. Jürgen Rühe, focussed on the production of small chips for measuring the flow of ions using membranes. This project was carried out in cooperation with Prof. Dr. Jan C. Behrends from the Institute of Physiology at the University of Freiburg. “It has taken almost exactly eight years from the time the Department of Microsystems Technology and the Department of Physiology started to work together until our company will be officially established in autumn 2013,” said Gerhard Baaken recalling the beginnings of the successful cooperation. The start-up company is funded through the EXIST programme run by the German Federal Ministry of Economics and Technology (BMWT). In addition to Baaken and Behrends, the Ionera team also includes biophysicist Dr. Ekaterina Zaitseva and engineer Dr. Sönke Petersen.
Baaken and his team’s invention relates to the measurement of ion conduction in tiny pores located in an artificial cell membrane. In fact, the pores can be used to accurately determine the size and composition of individual protein, DNA and polymer molecules. However, this can only be done if the molecules contained in the salt solution move into the pore. When a larger molecule moves through the pore, the pore is temporarily obstructed and the flow of ions decreases. “The degree of obstruction provides us with information about the size and composition of the molecules that are causing it,” Baaken explains.
Changes in electrical resistance are measured at the nanopores. As is the case with light barriers, the presence of a peptide can thus be detected in real time. The analyte, i.e. the particle of interest to the researchers, does not need to be charged. The only prerequisite is that it fits through the pore. Researchers usually use a toxin of the bacterium Staphylococcus aureus (alpha haemolysine) for such purposes. Alpha haemolysine is a complex protein that forms a transmembrane nanopore. It is integrated into the membrane of red blood cells in S. aureus infected individuals. Biological nanopores have the advantage that their conductance is relatively stable. In addition, they can easily be modified by mutation and chemical modification. Such modifications generate new analyte binding sites. “Of course, nanopores were not invented by us; they were invented by nature,” said Baaken pointing out that his team are simply making use of a tool that nature has already invented. So if not the nanopore itself, what is the invention that led to Ionera’s foundation?
The crux of nanopore analysis is the production of artificial lipid membranes (BLM: black lipid membranes) into which the pore is introduced. This is in fact a fairly tricky business. “Existing methods can only be carried out manually by specialists,” said the microsystems engineer.
A process known as painting, which is similar to the application of paint, involves the use of a Teflon spatula for applying an oil solution containing membrane components (phospholipids) on a small cavity opening, where the lipid-oil layer becomes thinner over time. This is a highly sensitive and time-consuming procedure, which, for reasons we never understand, sometimes works and sometimes does not. “There is always an element of voodoo involved,” said Baaken, smiling. “A BLM pioneer once told me that he was so frustrated with the procedure that he threw laboratory glassware at the wall. And I know of another BLM specialist who, when the bilayer did not form, told his students to remix all the ingredients and try again but, more importantly, to change their socks before repeating the process,” Baaken adds. In addition, the method is unpredictable and cannot be automated. In order to speed up the process, Baaken and his team have developed the MECA (microelectrode cavity array) chip, a platform of two square centimetres with an active centre consisting of 16 microelectrode cavities (MECs). Each cavity is 6-50 micrometres in diameter, eight micrometres deep and is equipped with an individually addressable microelectrode. The cavities are arranged in an array 100 to 200 micrometres from each other. The active centre is surrounded by contact areas, which are part of the microelectrode cavities and which transmit the ion current in the salt solution between the electrodes to the amplifier. Gerhard Baaken explains the most important features of the MECA chip: “We have developed a procedure that enables lipid bilayers to form automatically over each microcavity. And it is worth adding that this works by pressing a button.” If a membrane gets damaged, pressing a button will generate a new one. In contrast to previous methods, the use of remotely actuated painting is a huge step forward. Ionera’s patented SPREAD technology has a near 100% success rate in the formation of BLMs.
Ionera’s platform technology enables the rapid, fully automated and high-throughput electrical measurement of individual molecules in nanopores. Covered by a synthetic membrane, each cavity contains only one nanopore and produces a specific flow pattern. This tool is perfect for application in the pharmaceutical industry, although at present the prototype only contains 16 cavities. According to Baaken, the number of cavities can be increased at will. The chips can be used for high-throughput, label-free molecular analyses, the detection and characterisation of proteins and for carrying out single-molecule mass spectrometric analyses. “Our device is a generic tool that can be used for a broad range of different applications,” said Baaken, going on to add, “users decide what they want to use it for. We offer a plethora of different possibilities.” Bioelectric nanopore analyses are so attractive because they are extremely accurate, enable the efficient generation of data and save money and time. “DNA sequencing is the Holy Grail we are all after,” said Baaken highlighting that this seems to be within reach. “The different bases of single DNA strands look like a string of differently sized pearls. The varying sizes of the pearls clog the pore to a lesser or greater extent. This is the principle that enables us to determine the different bases of a DNA strand.”
The company will be officially established in autumn 2013 and many things still remain to be done. However, the most important courses have been set. While Ionera is specifically focussed on the production of the chips and the analysis kits, Nanion Technologies GmbH, which is a company previously spun out from Prof. Behrends’ laboratory, produces the necessary measurement devices. Another important focus of Ionera is on the development of assays, in particular chip layouts and kits for specific applications. Ionera’s MECA technology therefore has the potential to become a standard method in the field of biotechnology in a few years’ time.
Further information:Dr. Gerhard BaakenInstitute of Physiology IIHerman-Herder-Str.779102 FreiburgTel.: +49 (0)761/ 203-5145Fax: +49 (0)761/ 203-5191E-mail: gerhard.baaken(at)physiologie.uni-freiburg.de