Antibiotics have long been used as all-purpose weapons against infectious diseases – too often and too early, as we now know. This tendency has caused many bacteria to become resistant to standard antibiotics. The search for new substance classes has proved quite difficult. Care must therefore be taken to use existing antibiotics prudently in order to reduce the number of bacteria becoming resistant to them in the long term. Researchers from IMTEK and BIOSS at the University of Freiburg have jointly developed a sensor platform to simultaneously quantify several antibiotics in human blood within a few minutes. Therapy duration and dosage can thus be tailored to the requirements of individual patients. The biosensor is now being further developed for other medical diagnostics applications.
As we now know, people with identical diseases can react very differently to the same therapy: a drug dose that is too low and hence ineffective in one patient, might be too high and even lead to adverse effects in another. This difference is due to drug-metabolising enzyme variants that break down drugs either rapidly, slowly or not at all in different people. This also applies to the breakdown of antibiotics, which are additionally associated with the problem that the bacteria for which they are prescribed no longer respond to many standard antibiotics. The far too frequent and inappropriately dosed use of antibiotics has led in recent years to the bacteria that cause disease developing resistances to these drugs. Multi-resistant bacteria are increasingly responsible for the fact that life-threatening infections are difficult to treat with existing antibiotics. Intensive research is therefore being undertaken to find strategies that can prevent bacteria from developing antibiotic resistances in the long term.
In the Laboratory for Sensors (Director: Prof. Dr. Gerald Urban) in the University of Freiburg’s Department of Microsystems Engineering (IMTEK), Dr. Can Dincer and his research group have spent the past few years developing a biosensor for which Dr. Dincer, a microsystems engineer, won a Gips Schüle Young Scientist Award in January 2017. The sensor can be used to quantify various medical parameters in body fluids, including the concentration of antibiotics. The sensor platform is based on photo films that are layered like puff pastry, says Can Dincer.
”The biggest advantage of this fluidic system is that it is simple and inexpensive to manufacture and can be used in any laboratory without the need for additional equipment,” Dincer says. ”The system is made using a simple photolithography process that is used in laboratories around the world.” The biosensor system consists of relatively high (around 60 micrometres) microfluidic channels in which microfluidic analyses can take place. Different sensor formats with two, four and eight channels and traditional one-channel sensor systems are available, and are chosen in relation to the number of substances to be analysed in parallel.
The quantification of antibiotics takes place in the channels and is based on a method that Dincer’s team jointly developed with biotechnologist Prof. Dr. Wilfried Weber and his team from the BIOSS Centre for Biological Signalling Studies in Freiburg. The researchers’ antibiotic test system is based on a sensor system that occurs naturally in antibiotic-resistant bacteria. “The bacterial sensor system consists of a DNA sequence and a repressor protein. Imagine this constellation as a house with a guard dog,” explains Dincer. “When an antibiotic enters the cell, the repressor protein is removed from the DNA, making it accessible for transcription. In our test system, we use a specifically labelled protein that releases a signal telling us that an antibiotic has bound.” Analysis takes only 15 minutes, from sample to result.
The researchers demonstrated the applicability of the sensor platform with tetracycline and streptogramin antibiotics in human blood.1 “However, we are able to measure up to eight substances simultaneously,” says the scientist from Freiburg. “We are currently working on further improving the platform to make it suitable for measuring samples that are as small as possible in the shortest possible time. We currently have just the sensor but we also want to develop a suitable read-out system with micropumps, so that, ideally, we would be able to just add a single sample droplet (e.g. blood) and the machine would automatically come up with the result.”
In principle, the biosensor system can be used to measure practically any human body fluid/respiratory gas without requiring complex sample preparation - plasma, urine, respiratory gas and even viscous whole blood can be analysed. Therefore, the method has the potential to be used for a broad range of applications. “One possibility would be to use the sensor for measuring antibiotics concentrations in patient samples during surgery, thus enabling drug doses to be adjusted to the duration of the operation and the requirements of the individual patient,” says Dincer. “Such personalised therapies reduce the number of bacteria that become resistant to antibiotics and limit potential damage to the body.” The researchers have already assessed this possibility and the results are quite good, as Dincer reports.
The Freiburg team of researchers has also thought of using the biosensor to find out how quickly an individual organism metabolises an antibiotic in order to determine the appropriate dose for a particular patient. The sensor could also be used to determine the start and end of an antibiotics therapy. “We would then be able to determine exactly when antibiotics therapy should be terminated,” says the microsystems engineer. “Taking antibiotics for too long might lead to liver and kidney damage. Taking antibiotics for too short a period of time may promote the emergence of antibiotic-resistant bacteria. The new sensor could therefore pave the way to personalised antibiotics therapies.”
But it is not just antibiotics that can be analysed with the sensor platform. “This system can be used for many things,” says Dincer. The researchers will therefore soon be using the sensor for examining microRNA. “The microRNA expression ratio is strongly suspected of being predictive of a broad range of different diseases,” says Dincer. “We have received funding from the German Research Foundation to study this.” The manufacturing process of the chip is exactly the same, and the microRNA biosensor will look similar to the sensors used for personalised antibiotics therapies. The team already knows that the detection of microRNA works. Now, the IMTEK researchers are working on optimising the tests and perhaps finding a way to detect ribonucleic acids even faster than is currently possible, i.e. without involving PCR (polymerase chain reaction). “We are still at the very early stages. But we hope that in two to three years' time, we will be able to develop a completely new field of diagnostics,” concludes Dincer.
The scientists' major objective is to turn the sensor platform into a kind of plug-and-play toy for biochemists and doctors. Dincer comments: “Our principal objective is to create a portable system that works autonomously and that every patient could carry on them. This would make it possible to analyse substances over longer periods of time. However, before this becomes reality, many as yet unanswered questions, including ethical ones, have to be clarified.
1 André Kling, Claire Chatelle, Lucas Armbrecht, Edvina Qelibari, Jochen Kieninger, Can Dincer, Wilfried Weber, and Gerald Urban, Multianalyte antibiotic detection on an electrochemical microfluidic platform, 2016 Anal. Chem., 88(20), 10036 - 10043. DOI: 10.1021/acs.analchem.6b02294