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Nanosensor detects pathogens

Researchers from the Stuttgart-based Max Planck Institute of Solid State Research have succeeded in detecting tiny traces of DNA using sensors made from carbon nanotubes. The sensors are highly selective for specific DNA sequences and it is envisaged that they will be used for the rapid examination of blood samples.

The detection of DNA is required for the diagnosis of different diseases such as viral or microbial infections. The nanosensors developed by the group of researchers from Stuttgart are so sensitive that the time-consuming concentration and labelling of DNA is no longer necessary. The researchers have developed a routine method for the production of their nanosensors, which also enables the mass-production of the sensors. What the researchers have in mind is an analysis apparatus the size of a mobile phone that could be used in the field, for example in zones affected by epidemics.

DNA test using nanotubes: Researchers from the Max Planck Institute of Solid State Research immobilize single strands of DNA to the surface of the carbon tube. Matching DNA fragments (yellow) from the blood sample attach to these DNA strands and alter the conductivity of the nanotube. Changes in the conductivity are recorded using the two electrodes bridging the tube. © K. Balasubramanian

During the swine flu epidemics in 2009/2010, people presenting with a light cold or fever were put in isolation and tested for swine flu to confirm whether they actually had the disease or not. The long-term isolation that was necessary because there was no quick method to detect the disease caused patients considerable stress. A more rapid diagnosis for testing for infections, for example after an accident, would also be useful as it would enable suitable therapies to be put in place immediately.

However, it often takes days to detect a viral infection. This is because the pathogen’s genetic material (DNA or RNA) is present in the patient’s blood only at a very low concentration. In addition, the viral DNA/RNA cannot be detected using conventional analytical methods. Therefore, viral genetic material must be enriched to a level above the detection limit using a method known as polymerase chain reaction, or PCR for short. The amplified DNA is labelled with a fluorescent molecule that can subsequently be detected under a fluorescence microscope.
The sensor chip developed by a group of researchers led by Kannan Balasubramanian from the Max Planck Institute of Solid State Research does not require the DNA to be enriched. Instead of detecting the viral DNA using a microscope, the chip uses highly sensitive electronics. The core of the chip consists of carbon nanotubes (CNT) which themselves consist of a lattice of carbon atoms. The tubes, which are less than a nanometer in diameter (i.e. less than one millionth of a millimeter) are characterised by excellent conductivity. CNT are especially suitable as highly sensitive sensors because their electrical resistance changes abruptly if molecules attach to their surface. This can be explained by the structure of the nanotube, which does not have any atoms in the interior; all carbon atoms are located on the surface, forming a regular lattice. When a foreign molecule binds to one of the nanotube’s carbon atoms, it interrupts the lattice pattern at this spot, thereby disrupting the free passage of electrons over the surface of the carbon tube.

However, the sensitivity of the carbon tubes is only half the story. Since a biological liquid contains many DNA molecules in addition to the one that is being sought, the nanotube must be rather choosy. Scientists refer to this as selectivity, which means that the nanotubes need to be highly selective in detecting particular DNA sequences. The researchers are able to make the nanotubes selectively perceptive by attaching certain DNA molecules to them prior to the test. Single DNA strands attached to the nanotube will only bind to complementary DNA strands and form a double helix. The two complementary DNA stretches fit into each other like a key in a lock. When the two DNA molecules bind together, the presence of the second DNA molecule increases the nanotube’s resistance, i.e. the electron density on the carbon nanotube increases and hence its conductivity falls further.

Sensor chip for mobile diagnostic instruments

Sensor chip for the detection of pathogens: the chip is mounted on a plate. Pairs of platinum electrodes are bridged with nanotubes. The chip is covered with a poly (demethylsiloxane) plate (PDMS) with two reservoirs and one microchannel above the gap between the electrodes. The reservoirs and microchannel are filled with buffer solution. The reference electrode enables stable and reproducible measurements. The scanning force microscope (bottom) image shows the nanotubes in their position between the electrodes. © K. Balasubramanian / Applied Chemistry

The researchers from Stuttgart have constructed chips on which several of the pre-treated carbon nanotubes create a bridge between two electrodes. The electrodes form a channel just a few thousandth of a millimetre wide through which the solution under investigation flows and comes into contact with the nanotubes. The electrodes can be coupled to a measurement device that determines the change in conductivity.

The researchers used a chemical procedure to remove the DNA molecules from the nanotubes after measurement, thereby enabling the sensor to be reused. A reference electrode makes the measurement results reproducible and stable.

An initial experiment involving a solution of synthetic DNA molecules led to the identification of molecules in concentrations that were lower than any that had previously been detected by sensors. In figures, this means that the sensor detected 2000 molecules of the sought-after DNA in a 30 microlitre solution. This corresponds to a 100 attomolar DNA solution (one attomol corresponds to a billionth part of a billionth mole.)

“We believe that the method can be further optimised, enabling us to detect even lower concentrations, and in the best case even individual molecules,” said Kannan Balasubramanian. The next goal is to be able to investigate real biological liquids. Investigations into this issue have already started.

The team is funded by the German Ministry of Education and Research and its success is based on long-term research work which made it possible to develop a routine method for the production of highly sensitive sensor chips with carbon nanotubes. “This method can be scaled to any size,” said Kannan Balasubramanian, "which makes it possible to mass-produce the sensor chips". Balasubramanian therefore believes that the nanosensors produced by his team will at some stage in the future be used for diagnostic devices the size of mobile phones aimed at the quick and reliable identification of pathogens in the field or in hospitals.

Website address: https://www.gesundheitsindustrie-bw.de/en/article/press-release/nanosensor-detects-pathogens