Label-free cell analysis : an infrared sensor for CH2 - stretch ratio determination and a microfluidic cell-cell separator

Abstract

In this thesis two label-free cell analysis topics are being discussed. The main subject is an infrared absorbance based sensor system for the determination of the [CH tief 2]-symmetric to [CH tief 2]-antisymmetric stretch ratio. It was investigated whether the designed label-free measurement method of the sensor system can aid in the detection of the presence of cancer cells. The other topic is purification methods for cell samples from cell culture debris and bacteria contaminants. In particular the label-free separation method based on travelling wave dielectrophoresis (twDEP) was studied. The majority of tumour detection methods available to date are based on visual inspection of primary tumours, standard histopathology techniques applied on tumour biopsies using Haematoxylin & Eosin staining, and immunohistopathology using antibodies against tumour antigens. Visual inspection methods are based on morphological interpretation, and therefore subjective, discrepancies in the diagnosis have been reported.<br />Improved and objective diagnostic methods are highly important for increasing the overall survival rates of cancer patients. The infrared sensor system, which measures a potential diagnostic variable for the detection of tumour cells and tissues within a short time span, records IR absorbance of the [CH tief 2]-symmetric and [CH tief 2]-antisymmetric stretch at 3.51 um and 3.42 um, respectively. In addition, the IR absorbance at two reference wavelengths is determined to compensate for variations in the sample thickness and water content. The main components of the designed and realised sensor system are mid-IR emitting LEDs, a photodiode, and narrow band-pass filters. To investigate the potential of the realised sensor system, normal and various tumour cell lines originating from melanocytes and kidney epithelial are tested, both in dried condition and in suspension. The presented methodology yielded statistical significant [CH tief 2]-stretch ratio differences between the individual cell lines, normal and tumorous, of both epithelial kidney and melanocyte origin. Also, the mechanism influencing the [CH tief 2]-stretch ratio of mammalian cell membranes was investigated. A normal non-tumorous cell line was exposed to the plasma membrane bound cholesterol reducing agent methyl-[beta]-cyclodextrin. Measurement results indicate that an increase in [CH tief 2]-stretch ratio arises when there is a decrease in the membrane stabilizing agent cholesterol. The sensing method allows the discrimination between normal and tumour cells. In addition, the method shows high potential for improvement of staging of suspicious tissues and can easily be adapted in a point-of-care instrument. A microfluidic twDEP separator is designed and realised to separate specific cells from a mixture. Viable suspended-grown Jurkat cells were successfully separated from cell culture debris and L. casei bacteria. In conclusion it is shown that label-free techniques can be applied to determine the presence of tumour cells and to separate or sort specific cells from a cell mixture. These methods promise simpler and more cost-effective analysis techniques that can be applied in diagnostic instrumentation and optimisation of curing therapies.<br />