A MIP, molecularly imprinted polymer based impedimetric sensor for the detection of small MW molecules

Abstract

Currently there is an increasing demand for sensors for medical, environmental and industrial applications with strong requirements concerning selectivity, sensitivity and selectivity. Ordinary biosensors make use of biological systems, such as antibodies, enzymes, cells, etc., as recognition element. These biomolecules posses a high selectivity and affinity, however they are not available for any target molecule and are not resistant against hostile environments, e.g. high temperatures, high and low pH or organic solvents. As proof of principle, a MIP, molecularly imprinted polymer, based sensor for L-nicotine, C10H14N2, has been developed. Furthermore, the first step was made for detecting a more medical relevant molecule, histamine, a mediator. The imprinting technique is based on the development of a non-covalent complexes formed between the target molecule and suitable functional monomers, being methacrylate (MAA). Subsequently cross-linker, ethylglycol dimethacrylate (EGDM) is added to this mixture to form a matrix in which the complexes are fixed. After extraction of the target molecule from this matrix, a tailor-made highly selective synthetic receptor is created which can rebind the anlyte according to its shape and functionality. For further processing the bulk monolith of the polymer is mechanically grinded to small micro-particles. Besides the MIP, a non-imprinted polymer, NIP, is synthesized. This acts as a reference material to be able to distinguish non-specific adsorption of the target to the surface of the MIP or NIP. Optical batch-rebinding experiments showed specific binding of nicotine and a clear difference could be made between L-nicotine en L-cotinine, the oxidizedform of nicotine, which only differs one oxygen atom. Due to the heterogeneous character of the binding sites of the MIP the Freundlich isotherm was used to model the binding characteristics. To obtain a sensing device the micro particles were glued on a metal electrode using a conjugated polymer OC1C10-PPV, allowing electronic detection based on the impedance principle. The surface coverage was investigated using optical microscopy and scanning electron microscopy. The coverage of the samples was about 20%. As a second reference technique fluorescence spectroscopy was used. The idea was, when the binding site would be close enough to the conjugated polymer chain the fluorescence of the conjugated polymer would be quenched. However, the measurements revealed no indication whether L-nicotine or L-cotinine could quench the fluorescence. Impedance spectroscopy is used to obtain an electronic read out. By measuring the impedance in the lower frequency range, below 1 kHz, small changes at the interface of the electrode and the solution can be visualized. The impedance is measured time-resolved for four different channels inside the measurement device. The measurements are carried using an impedance analyzer. Also, the first step is made towards a hand-held device. A apparatus was developed which could track impedance changes of eight different channels at a fixed frequency. Upon introduction of L-nicotine an impedance increase was visualized. This increase was also visible for the reference channels, NIP and blank polymer, but here the rise was less. This augmentation is probably due to non-specific adsorption at the surface of the material. The impedance sensor shows to be very sensitive for the target molecule L-nicotine and insensitive for the resembling molecule L-cotinine. The response time was only a few minutes with a detection limit of 0.3 ng/ml within a linear range of 2 to 5 nM for the L-nicotine MIP-based sensor. The gravimetric principle can also be applied to investigated the binding properties of the MIP sensor. A quartz crystal is functionalized in the same way as with the impedance sensing technique. In this way a microbalance is obtained. The resonance frequency of the quartz is dependent on the massloading of the surface. When L-nicotine is bound to the MIP a frequency decrease is visualized. In this way a MIP based quartz crystal microbalance, QCM, was created which was sensitive for the presence of L-nicotine, while showing little affinity for the resembling L-cotinine. The NIP based QCM sensor showed a lower affinity for both molecules. These results confirmed the conclusion from the impedance measurements. This method allowed also for the possibility of flushing the measurement cell. This showed the possibility of regeneration the MIP sensor when flushed with distilled water. The first tests were also performed towards a more medical relevant sensor. A histamine MIP was used instead of the L-nicotine MIP. Histamine is a molecule which is involved in different biochemical processes. For example it plays an important role in the gastrointestinal tract, where it is in indicator for stress. This sensor showed a higher affinity compared to the L-nicotine sensor. This project initiated the further research towards a MIP based impedimetric sensor. Using a relative easy method, a cheap and fast sensor was developed with high sensitivity, enabling it to measure small molecules for pharmaceutical, environmental, diagnostic or biotechnical applications. The suggestion for further work in this field is therefore, trying to develop a MIP based impedimetric sensor for molecules with a large field of applications. Next, the miniaturization of the sensor has to be further investigated making the step to a hand-held device.