Rapid pathogen detection from a single DNA molecule

With resistance to antibiotics on the rise worldwide, German researchers have developed a process for rapidly detecting multidrug-resistant pathogens. The standout feature of their platform? Just a single molecule of DNA is sufficient for pathogen detection.

Choosing the correct antibiotic to treat bacterial infections is a deciding factor when it comes to the success of a treatment — and it is particularly difficult to do so in cases where a disease is caused by multidrug-resistant pathogens, which are unaffected by many antibiotics. Furthermore, searching for the most effective antibiotic often requires information about the bacteria’s genome — information which is not readily available at medical practices and can only be obtained through a laboratory diagnosis.

To accelerate and simplify the process, researchers from the Fraunhofer Institute for Physical Measurement Techniques IPM collaborated with the Ludwig Maximilian University of Munich to develop a new platform for detecting pathogens on the basis of single molecules on a microfluidic chip. The focus of the SiBoF (signal boosters for fluorescence assays in molecular diagnostics) project, funded by the German Federal Ministry of Education and Research (BMBF), lies on an easy-to-use point-of-care (POC) detection method.

The portable, compact test platform is equipped with an automated fluidic system; all necessary reagents are stored within the system. The injection-moulded microfluidic chip is incorporated in a drawer in the test system, where it is supplied with the reagents through the fluidics system before the optical analysis takes place.

“We detect part of the pathogen’s DNA strand,” said Fraunhofer IPM’s Dr Benedikt Hauer, who serves as project manager. “Using our new process, even a single molecule of DNA that binds to a specific site on the microfluidic chip is sufficient to do this. Fluidic channels are integrated into the chip, the surfaces of which are primed with binding sites for specific pathogens.”

Typically, target DNA molecules are detected by means of specific fluorescence markers. A unique feature of the new method is that researchers are utilising antennas with nanometre-sized beads, which amplify the optical signals of these markers. Because of this, chemical amplification via polymerase chain reaction (PCR) is not required.

“The optical antennas consist of nanometre-sized metal particles that concentrate light in a tiny region and also help to emit the light — much as macroscopic antennas do with radio waves,” Dr Hauer said. “These metal particles are chemically bound to the surface of the chip.”

A structure of DNA molecules, known as ‘DNA origami’ and designed by the Ludwig Maximilian University of Munich, holds both of the gold nanoparticles in place. Between these nanoparticles, the structure provides a binding site for the respective target molecule and a fluorescence marker. This patented design provides the basis for the novel assay technology.

“The particles, which are 100 nm in size, serve as antennas,’ Dr Hauer said. “Field enhancement, caused by plasmonic effects, takes place in the hotspot between the two gold particles. If a fluorescent dye is placed there, the detectable long-wave fluorescence radiation is enhanced multiple times. Using this method, a single molecule can be detected using a small, compact optical device.” Low concentrations of pathogens can thus be detected.

The result is available after one hour and is displayed on the monitor. This is true not only for multidrug-resistant pathogens, but for any type of DNA molecule. In principle, the single molecule assay can be adapted to molecules beyond DNA, such as RNA, antibodies, antigens or enzymes. Numerous tests have successfully confirmed the functionality of the process.

At the heart of the POC device is a miniaturised high-resolution fluorescence microscope, developed by Fraunhofer IPM. Specifically developed image analysis software identifies single molecules and by doing so enables the captured target molecules to be counted, providing a quantitative result. The fluorescence is stimulated using LEDs, which are affixed underneath the cartridge containing the fluidic channels.

The POC system will be presented at the MEDICA 2021 trade fair in Düsseldorf from 15–18 November. In future, the platform could be introduced as part of POC diagnostics on hospital wards or in medical practices — either as an alternative to the established PCR analyses or in combination with other diagnostic methods.

Image caption: The compact device for detecting multidrug-resistant pathogens performs all stages of the reaction automatically and provides a result within one hour. Image ©Fraunhofer IPM.

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