New ideas for improving diagnostics

Whether with glowing nanotubes or molecular tools - new diagnostic methods could deliver quick results and be available anywhere in the world.

Prof. Dr. Sebastian Kruss, Ruhr-Universität Bochum, has other plans. He wants to detect the presence of viruses like Sars-CoV-2 or bacteria by carrying out an investigation actually the body of the infected person or directly in blood or saliva samples – quickly and easily; no need for time-consuming sample preparation.

His research is still at the basic stage. But if Kruss and the other physicists, chemists and life scientists involved can keep up the good progress they have made so far, there will soon be unimagined new possibilities in diagnostics – thanks to tiny, luminous nanotubes. How does the method work, and where could it be used?

Sensors strike when viruses fall into "the trap"

At the heart of the new detection method are tiny carbon tubes of less than one nanometer in diameter. When these nanotubes, which can be obtained by burning methane, for example, are irradiated with light, they begin to glow. The human eye cannot see this glow because it is in the "near-infrared" and not in the visible light wavelength. However, special cameras can "read" the light signal that emanates from the thin tubes.

The nanotubes are the luminous components of a sensor. "Crucial to its function, however, is the organic phase around the tube, which can consist of antibodies or other detection molecules, for example," says Sebastian Kruss. The tubes are thus surrounded by a kind of "molecular adhesive layer" that can selectively recognize and bind viruses, bacteria or even their metabolic products. 

A nanosensor on the artificial joint, however, would immediately "strike" as soon as unwanted bacteria begin to spread. "The advantage of the technology is that the nanotubes glow in the near-infrared range, a range that remains virtually undisturbed by background signals emanating from the surrounding tissue," says Sebastian Kruss. The optical signal can penetrate body tissue up to one centimeter because it is hardly absorbed or scattered by water, cells or blood. A camera could then "look" through the intact skin into the body and capture the signal sent by the sensors.

Kruss is currently working on a prototype of such a camera with a team at the Fraunhofer Institute for Microelectronic Systems and Circuits in Duisburg. In principle, however, the light signal could also be captured by a cell phone camera. This application on a cell phone might sound a bit like science fiction, but it is not so improbable that one day we could find out whether or not we are infected with Sars-CoV-2 or other pathogens with the help of a corresponding nanotube test kit and an app on our smartphone.

Diagnostics for all – LAMP-PCR

Anyone in this country who orders the molecular ingredients for the laboratory detection of viruses or bacteria usually receives the enzymes and reagents they need the very next day. "If, on the other hand, colleagues at our partner university in Addis Ababa request these test enzymes from the manufacturers, they usually have to wait one to two months for them and pay three to four times the price," says Johannes Kabisch, a professor at Darmstadt Technical University. In a cooperative project with researchers at the University of Cambridge and the Ethiopian Biotechnology Institute, Kabisch is working to address this disgraceful state of affairs: How can the molecular tools needed for viral diagnostics, for example, be produced in countries with scarce resources? "We want to take the technology to where it is needed," says the biotechnologist, outlining one of the project's core ideas.

Until now, however, LAMP PCR was clearly inferior to the classical variant. It was not as robust and not as sensitive. The method has since been improved by researchers worldwide. "LAMP PCR gives a reliable yes or no answer, indicating whether the sample contains the suspected pathogen or not," Kabisch says. In future, the technique could become a good alternative to the classical PCR method. However, it does not provide exact information about the amount of viruses or bacteria contained.

Bioproduced enzymes are key

The enzymes needed for LAMP PCR are produced by bacteria of the Bacillus subtilis variety. The genetic material of these bacteria is genetically modified to produce the enzymes "reverse transcriptase" or a "polymerase".

...the bacteria
do all the work

The first enzyme is needed for the detection of RNA viruses such as Sars-CoV-2. It translates RNA into DNA, which can then be amplified with the second enzyme, a kind of molecular copier, and thus made visible. Bacteria also produce these enzymes in a conventional production process. However, they are then purified from the disrupted bacterial cells. "In the production method we have chosen, the bacteria do all the work," says Johannes Kabisch. They make the enzymes and are then induced to enter a steady state, the bacterial spore, by altering culture conditions. The key feature of this technique is that "the spore carries the desired enzyme on its surface, it even kills its mother cell itself, and a simple centrifugation is all that is needed to purify the enzymes attached to the spores," says the researcher from Darmstadt.

The enzymes are not only easier to purify and cheaper to produce, they are also more stable because they are attached to the spores and last for about three months even at room temperature. However, before enzymes derived from such bioproduction arrive in diagnostics, all sorts of fine-tuning work must be done, test protocols must be checked and found to be reliable. Kabisch is confident: "By carefully adjusting the production process and testing the obtained enzymes, we will be able to develop reliable test systems". Following a successful transfer of knowledge from Darmstadt and Cambridge, he hopes to be able to produce enough enzymes in Addis Ababa for 10,000 prototypes of a Sars-CoV-2 test kit in the not-too-distant future.