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“Development of biosensors for the detection of histamine for intestinal application.”  
In recent health care is a growing need for bio- and chemosensors for the rapid and accurate detection of molecules. A biosensor uses a recognition element of biological origin. Immunosensors in particular are biosensors that use antibodies or immunoglobulines as their biological recognition element. Sensing systems based on biological recognition elements have some restrictions, limiting their use. Replacing the biological recognition element by a chemical receptor is therefore becoming of increasing interest. As opposed to the sensors containing biological recognition elements, biomimetic sensors, containing artificial receptors, are chemically and physically inert. Using Molecularly Imprinted Polymers (MIPs) the specificity and affinity of biological receptors can be mimicked. In addition, a MIP-based sensor can measure in harsh environments.This is beneficial for the use of sensors in vivo, in bodily fluids or in the intestines, for example in research for the Irritable Bowel Syndrome (IBS).

The IBS is characterized by visceral hypersensitivity. The pathogenesis is poorly understood, but there is evidence that mast cells are involved in this process.Mast cells degranulate upon activation, and release histamine, tryptase and other compounds. The accessibility of the intestine makes it difficult to measure this intestinal mast cell activation in vivo. Therefore a biosensor for the detection of histamine and tryptase in vivo in the intestine is developed.

Firstly, prototype immunosensors for the rapid and accurate detection of histamine and tryptase are constructed. The different building blocks of this biosensor are under investigation: AFM and contact angle measurements confirm the electrode functionalization with biological receptors. Antibodies against tryptase and histamine were successfully immobilized on polymer-based electrodes, incubation of 100 pmol/ml antibodies result in saturated surfaces. It is shown that physical adsorption is a fast and easy way to build a recognition layer. The sensor detects the antigen impedimetrically. For tryptase (135 kDa) coplanar electrodes are coated with the polymer MDMO-PPV and antibodies are immobilized on the polymer surface using physical adsorption. Binding of these 12 antibodies with their specific antigen will result in a measurable change in capacitive properties at the interface. The immunosensor shows a 10% response to 100 pmol/ml tryptase. Low-molecular weight molecules can for the first time also be impedimetrically detected with a direct assay by using IDE’s. For the detection of the antigen histamine (111 Da) IDE’s functionalized with antibodies against histamine are used to detect 50 nmol/ml histamine with a 10% change in the impedimetric signal. IDE’s are proven to be beneficial for use in future electronic sensing of tryptase and histamine.

Biomimetic sensors based on molecular imprinted polymers (MIPs) can be an alternative to sensors with chemically and physically instable biological receptors. Molecular imprinting leads to the formation of inert polymer particles with nanocavities, which mimick the selectivity and specificity of biological receptors such as natural antibodies. Histamine occurs in harsh environments in food and bodily fluids. MIPs can withstand such harsh environments. It is demonstrated that MIPs can be readily incorporated into a biomimetic sensor for the detection of histamine in aqueous media. Using electrochemical impedance spectroscopy, histamine is successfully detected in the nanomolar range. In pH neutral environments the sensitivity is 45% to 10nM histamine. Typical physiological conditions in i.e. mast cells are around 200 nM. A dose response curve is measured in the 0-12 nM range. Sensor saturation begins at 9.3 nmol/l. Using the analogous molecule histidine, it is demonstrated that the impedimetric sensor is specific for the detection of histamine.

When a sensor for the detection of histamine in bodily fluids or in the intestines is developed the pH of the environment is an important factor to consider. Although the MIP can withstand a wide range of pH values, the pH of the electrolyte affects protonation or deprotonation of target molecule and MIP. The sensor is tested under various pH environments between pH 5 and pH 11. These measurements have shown that this pH dependent degree of protonation of both the MIP and histamine has a substantial impact on the formation of hydrogen bonds, which are needed for binding of the target molecule to the nanocavity in the MIP. Hence, the detection of histamine by a MIP-based sensor is affected by the pH of the solution. A novel model has been established to verify the impedimetric results. This model is useful for adapting the MIP to allow for the 13 formation of hydrogen bonds at lower pH values. This makes it possible for histamine to be impedimetrically detected at low pH conditions by means of synthetic receptors.