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The detection of inflammation in “single-use laboratories“

Depression, rheumatism, sickle cell anaemia – elevated levels of substance P in blood plasma might be an alarm signal. The molecule plays an important role in generating an inflammatory response. Commercially available tests involving microtitre plates are time-consuming and expensive. Dr. Hüseyin Bakirci and his team from the Department of Microsystems Engineering (IMTEK) at the University of Freiburg have developed a chip the size of a fingertip that enables the cheap, fast and highly sensitive quantitative measurement of substance P and other biomarkers in patient samples.

The discovery of the small peptide was a rather painful affair. Substance P is a neurotransmitter consisting of eleven amino acids. It transfers signals between nerve cells when pain occurs. However, the molecule is also a key mediator of inflammatory reactions of the immune system; it is released from immune cells during immune responses to bacterial invasion and attracts further immune cells to the site of infection. This can cause the immune system to overreact and pathological symptoms can develop. The peptide has been shown to be associated with rheumatism and inflammatory diseases of the nervous system as well as psychiatric disorders such as depression and anxiety. The presence of substance P in the blood or saliva of patients can thus provide doctors with information about the causes of disease symptoms. “Clinicians and industry are all particularly interested in handy systems for the rapid and reliable measurement of biomarkers such as substance P in body fluids,” said Dr. Hüseyin Bakirci from the Department of Microsystems Engineering (IMTEK) at the University of Freiburg.

Five men standing in front of a building.
Team from the IMTEK’s Laboratory for Sensors in Freiburg: (from left to right) Prof. Dr. Gerald Urban (head of department), Dr. Hüseyin Bakirci (head of Bioanalytical Microsystems), Dipl.-Ing. Josef Horak, Dipl.-Ing. Can Dincer and Dipl.-Ing. Jochen Kieninger (head of Sensor Technology). © Dr. Hüseyin Bakirci

A thing of the past: waiting for two days for the results

Dr. Bakirci is head of a group of researchers in the Laboratory for Sensors directed by Prof. Dr. Gerald Urban. Bakirci and his team have developed a plastic film the size of a fingertip. The team’s miniaturised sensor system looks rather like the litmus paper that turns red when immersed in acid. However on closer inspection, the plastic film is in fact a labyrinth of microscopic channels and several platinum electrodes, and is in principle a miniaturised laboratory. “When I joined Prof. Urban’s group around three years ago, the researchers were part of a large network project focusing on the transfer of immunoassays for the detection of specific biomarkers from microtitre plates to small chips,” said Dr. Bakirci who was tasked with establishing the principle of immunoassays in a microscopically small system of capillaries and coupling it with an electrochemical sensor system. The goal was to fit the detection system on the aforementioned small plastic film and make it cost-efficient to produce.

Lab-on-a-chip developed by the Bioanalytical Microsystems team at Freiburg-based IMTEK. The chip enables the quantitative detection of neuromarkers such as substance P within 1.5 hours. © Dr. Hüseyin Bakirci

The microsystems engineers at IMTEK are now able to detect ten picogrammes of substance P per gramme of blood plasma, the same high level of sensitivity as commercially available microtitre plate test systems. However, the new chip is much cheaper. The polyimide material used for the chip is cheap and the amount of sample material can be reduced considerably. In addition to all these advantages, the miniaturised test system only takes 1.5 hours instead of two days to produce results and is thus considerably faster than traditional ELISA tests involving microtitre plates. This is perfect for hospital doctors who are concerned with rapid results, and particularly in outpatient settings.

Migraine, epilepsy and doping in competitive cycling

The principle of the competitive immunoassay is fairly straightforward: capture antibodies are immobilised on the surface of the sensor’s capillary channels. These molecules bind to substance P contained in the patient sample (blood serum, blood plasma or saliva) that is injected into the capillary system. In addition to the patient sample, a defined concentration of modified substance P molecules (labelled with the enzyme glucose oxidase (GOD) which catalyses the oxidation of glucose to hydrogen peroxide) is injected into the capillaries. The GOD-labelled and patient-derived molecules then compete for antibody binding sites. The larger the number of substance P molecules in the sample, the fewer labelled molecules are able to bind to the capture antibodies. Glucose is added to the solution and is oxidised by the GOD enzyme. This leads to the generation of hydrogen peroxide which then reacts with the electrodes that reach into the capillaries. The electrochemical reaction at the electrodes’ platinum surface generates measurable electrical current and provides information about the amount of substance P in the patient sample.

“The most difficult step in the development of this chip-based measurement system was the establishment of a method that enabled the reproducible immobilisation of the antibodies in the capillaries,” said Dr. Bakirci. The researchers also had to test the different characteristics of the capillaries in order to optimise parameters such as migration speed of the reaction fluid and incubation time required. The overall goal was to establish a system that required the user to simply add the sample and wait for 1.5 hours for the results to come up on a screen.

The fields of application for such a technology are fairly obvious. In addition to clinical applications for which Dr. Bakirci and his team are already developing biomarker tests for the diagnosis of meningitis, migraine, cardiac diseases and epilepsy, the lab-on-a-chip also has the potential to be used in mobile doping tests, for example in competitive cycling. Dr. Bakirci and his team are aiming to automate the system in order to make all the work steps as uncomplicated and standardised as possible without requiring too many working hours. Any new biomarker test method needs to undergo thorough validation and testing, which means the researchers need to involve industrial partners. Bakirci and his team at the IMTEK are working closely with a number of companies and expect to be able to market the substance P chip and potentially also other test methods soon.

Further information:
Dr. Hüseyin Bakirci, PhD
Head of Bioanalytical Microsystems
University of Freiburg
IMTEK – Department of Microsystems Engineering
Laboratory for Sensors
Georges-Köhler-Allee 103
79110 Freiburg
Tel.: +49 761/ 203 7264
Fax: +49 761/ 203 7262
E-mail: huseyin.bakirci(at)imtek.de

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/the-detection-of-inflammation-in-single-use-laboratories