Logo UHasselt

menu

IMO - IMOMEC


Search

IMO - IMOMEC

Logo UHasselt Universiteit Hasselt - Knowledge in action

THESIS DR. JAN ALENUS - SYNOPSIS

"QCM-based biomimetic sensors for the detection of nicotine, histamine and malachite green in body fluids and environmental samples."

The need for fast monitoring of compounds is increasing in medicine, food safety and environmental safety. This can be accomplished with the use of sensors which are highly sensitive and selective. Biosensors can fulfill these requirements with an array of different natural recognition elements such as DNA, antibodies, enzymes, cells, etc. The biggest concerns about these sensors are the cost, shelf life and their inability to be used in extreme pH or temperature environments. Synthetic recognition elements can achieve the criteria of the biosensors. However, without their major drawbacks. Molecular imprinted polymers (MIP) are a great candidate as a recognition element. Sensors that use synthetic recognition element are called biomimetic sensors. In this work, biomimetic sensors are created that use MIPs for recognition of histamine, malachite green and L-nicotine.


The principle of a MIP is the creation of a mold around a target molecule. After removal of this target, the mold can only rebind that target. To achieve this, a target molecule is mixed together with functional monomers (e.g. methacrylic acid) and crosslinking monomers (e.g. ethylene glycol dimethacrylate) in a fluid. The functional monomers will form connections with the target molecules. The cross linking monomers are then polymerized so the functional monomers are held into place in an established position. This bulk polymer is crushed and sieved in order to obtain small micro particles. When the target molecule is extracted from these particles, a custom made synthetic recognition element is achieved which can rebind its target molecule. For reasons of control, a non imprinted polymer (NIP) is created in the same way save the use of the target molecules thus creating a NIP which only will bind in a non specific way.
The effectiveness of the created MIP is first tested using (Ultraviolet/Visible) UV/Vis spectroscopy. MIPs are mixed with a solution containing a known concentration of the target molecule. The MIPs will bind a certain amount of the target molecule present. The MIPs (and thus the target molecule) are then removed from the solution and the remaining target molecules are measured with UV/Vis spectroscopy. This result is compared with a previously measured baseline, so the bound concentration of target molecule is known. UV/Vis is a reliable technique for preliminary testing. However, it can only measure in relative high concentrations (miliMolar range). The NIP is tested with the same protocol. Besides the NIP as a control for sensitivity, other similar compounds are used as selectivity control. Histidine is an amino acid from which histamine is metabolized and is thus an excellent control. Cotinine is the metabolite from L-nicotine and is also used as a control.


Once the MIPs is optimized for their target molecules, they are implemented into a sensor setup. In this work, impedance spectroscopy and gravimetric detection via a quartz crystal microbalance are used. Implementation of the MIPs occurs via immobilization on a polymer layer which was applied on the electrode surface. The polymer used is dependent on the read out technique. With impedance spectroscopy the conjugated polymer OC1C10-PPV is used while the Quartz crystals used for gravimetric detection are coated with PVC (polyvinyl chloride).


Impedance spectroscopy is a very sensitive technique that measures changes in impedance (electrical resistance) in a frequency spectrum over time. Measurements are performed in the lower frequency ranges as binding of the target molecules to the MIP induces capacitive changes in the recognition layer. It is possible to measure up to 4 different channels, which allows for differential measurements. Histamine measurements were performed in PBS (phosphate buffered saline) in the nanomolar range. Upon binding of histamine to the MIP, the impedance increased significantly while the NIP showed no change. Histidine also showed no impedance change, proving that the MIP is very selective towards histamine. After these successful measurements in PBS, tune brine was spiked with histamine and measured in PBS. Despite the complex fluid, the MIP was able to selectively bind histamine while the NIP showed no significant response. Malachite green was also successfully detected in the nanomolar range while the NIP showed a lower response.


Gravimetric detection uses a quartz crystal that vibrates at a certain predetermined frequency, the resonance frequency. When the target molecule binds to the MIP, the resonance frequency will decrease linearly with the added mass. This sensor setup is used to measure histamine in deionized water in the micromolar range. The MIP was able to bind 4 times the amount of histamine than the NIP. Malachite green was also successfully tested with QCM in the nanomolar range, which is low for the QCM read out technique. These measurements were performed in water with a pH value of pH 3. L-nicotine was measured with QCM-D (quartz crystal microbalance – dissipation). Dissipation is the amount of energy the crystal dissipates after a crystal excitation. The dissipation will change when softer material is bound to the crystal surface. Measurements were performed in deionized water and in PBS. The amount of L-nicotine that can be bound in deionized water is greater than in PBS. This is due to the ions present in the PBS as they inhibit the interactions between L-nicotine and the MIP. Lower concentrations of PBS showed an increase in the binding potential of the MIP towards L-nicotine. When measured in PBS, the optimal pH value was pH 9. The dissipation changes of the MIP upon binding of L-nicotine were also significantly higher than the NIP in deionized water and PBS. The L-nicotine MIP was also tested with spiked urine and saliva. These test showed that the MIP was able to bind L-nicotine despite being in a complex matrix. As a final test, the saliva of a subject who chewed nicotine gum (4 and 2 mg L-nicotine) and chewing tobacco was collected and measured with the QCM-D. The MIP was able to differentiate between the 4 mg and 2 mg saliva sample. The MIP was also successful in binding the L-nicotine from the chewing tobacco samples.


This work has proven that molecular imprinted polymers are promising candidates for future sensor applications. The MIP was able to function in various complex matrices without losing its sensitivity. Further investigation could implement the MIPs in other more sensitive read out techniques that allow for easier miniaturization, creating a hand held diagnostic tool for the future..