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"Molecularly imprinted polymers: synthetic receptors for diagnosticmedical devices."

Irritable Bowel Syndrome (IBS) is a functional bowel disorder which is characterized by abdominal pain and changes in bowel habit. It has a profound negative impact on the quality of a patient’s life and is associated with more than €20 billion of indirect and direct medial costs per year. Most IBS therapies are based on global relief of the symptoms, since the exact organic cause of the disease remains unknown. Recently, the involvement of biogenic amines such as serotonin and histamine has been postulated. The established technologies to determine the concentration of these target molecules have several disadvantages. The methods are costly, lack in speed, and require a lab-environment and sophisticated equipment. Therefore, the aim of this thesis is to develop a polymer-type sensor for the detection of histamine and serotonin for intestinal applications. The sensor platform should meet the following criteria: low-cost, fast response time, specific recognition in biological samples, and the possibility of measuring in vivo.

Molecularly Imprinted Polymers (MIPs) were used as polymer-type receptors since they are robust, can be produced at low-cost, and have a high affinity for their template molecules. They were synthesized by bulkpolymerization and subsequently ground to obtain a powder. Next, aluminum electrodes were functionalized with MIPs by thermal treatment. The particles were then integrated into a sensor platform which can specifically detect small molecules by two read-out technologies. The first technique is based on electrochemical impedance spectroscopy, the second on heat-transfer resistance.

First, a MIP for the specific detection of serotonin was developed. Various MIPs were synthesized and the MIP with the highest affinity for serotonin was selected by optical batch-rebinding experiments. The particles were then integrated in an open impedimetric sensor setup and a dose-response curve was measured in buffer solutions. For biological samples, a refined sensor-cell was developed. This flow-through cell was closed, ensuring safe-administration of patient’s samples, and featured an integrated temperature unit which improves stabilization and control over the system. With this setup, native serotonin concentrations in human blood plasma were determined. The obtained results were nicely in agreement with High Performance Liquid Chromatography (HPLC) reference tests. Furthermore, it was demonstrated that the impedimetric response upon binding of serotonin can be attributed to a capacitive effect at the interface between the MIP particles and fluid layer.

Histamine is the other biogenic amine of interest in this thesis. In previous work, histamine concentrations in buffer solutions ranging from pH 7-9 were determined with impedimetric read-out. For diagnostic applications, this target molecule should be detectable in a wider pH range as is it mostly occurs in mildly acidic environments. To understand the pH-dependent response of the MIP sensor, we proposed a statistical binding analysis model. With this model, we predicted the theoretical performance of a MIP based on the monomer acrylic acid in the required pH regime. The results were verified experimentally by UV-vis spectroscopy, microgravimetry and impedance spectroscopy. Histamine could be detected with impedimetric read-out in the physiologically relevant nanomolar concentration range in neutral and in mildly acidic buffer solutions.  As last validation step, this platform was used to analyze histamine concentration of mildly acidic bowel fluid samples of several test persons. We showed that this sensor provides reliable data in the relevant concentration regime, which was validated independently by Enzyme-Linked Immunosorbent Assay (ELISA) tests.

The electronic method requires minimal instrumentation, can perform fast measurements and is specific enough to determine concentrations in the physiologically relevant regime. We also presented an alternative read-out technique, the heat-transfer method (HTM), which is even more straightforward as it requires only two thermometers, an adjustable heat-source and a Proportional-Integeral-Derivative (PID) controller. The principle works as follows; upon rebinding of a target molecule, the heat flux through the nanocavity in the MIP is strongly reduced due to the presence of the template. As a result, the total heat transfer resistance (Rth) will be increased with the effect size being dependent on the amount of target molecule that is bound. For proof-of-principle purposes, aluminum electrodes were functionalized with MIP particles and L-nicotine measurements were performed in buffer solutions. The main focus of this thesis lies on serotonin and histamine; therefore dose-response curves in buffer solutions were also constructed for these target molecules. The achieved detection limits are comparable as with impedance spectroscopy. This technique was measured simultaneously with the Rth and provided a direct validaton of the results. The detection limit is in the physiologically relevant range, but application in biological samples was not extensively evaluated yet. As a first proof-of-application, measurements were performed on saliva samples spiked with L-nicotine. A dose-response curve could be constructed, showing the applicability even in complex matrices.
Both techniques are specific, low-cost and fast, thereby fulfilling the specified criteria to a great extent. The last criterium, the in vivo measuring for intestinal applications, imposes some challenges. To overcome these problems, the sample preparation needs to be altered possibily by directly polymerizing the MIPs onto the surface of the electrodes. The final step is to enter the industrial market of diagnostics. For this purpose, some future MIP platform designs were suggested. There are two more aspects we have to consider, before thinking of commercialization of the product:

i) A clinical trial should be performed to study a larger amount of biological samples and, additional patient samples. The latter is indispensable to determine if the technique is sensitive enough to detect occurring aberrations.
ii) The sensor platform should be transformed to an array format which enables the simultaneous detection of a variety of targets.

If we take into the account the speed, low-cost, and high specificity of the developed techniques, they can be considered high potential proof-of-principle technologies. These techniques could also mean a first step in the direction of incorporating MIPs into sensing devices for in vivo purposes. Measurements performed directly in the gastro-intestinal tract could provide a useful insight into the functioning of serotonin and histamine in the bowels. If the role of these biogenic amines further elucidated, it will open a new chapter in the treatment of IBS patients.