Design, implementation and characterization of an optoelectronic platform for the detection of immunosuppressants in transplanted patients by means of a microfluidic optical chip

The past few years have seen great interest and advances in scientific research on fluorescence-based microfluidic optical sensors for clinical/medical applications. In particular, the growing demand for point-of-care-testing (POCT) devices to be applied near the patient’s bed, has driven the development of simple to use, reliable and low cost microfluidic optical platforms. The main objective of the research project presented in this thesis is the development of a novel fluorescence-based microfluidic optical chip for the simultaneous analysis of different analytes and its integration into a stand-alone POCT device. The work was undertaken in the framework of the EU project NANODEM (NANOphotonic Device for Multiple therapeutic drug monitoring - FP7-ICT-2011) that aims at developing a POCT device for the measurement of immunosuppressants in transplanted patients, characterized by a narrow therapeutic range and serious potential side effects. The benefit of this device will be an optimized dosage of the therapeutic drugs to support patient management in a clinical environment. In particular, the system will accurately measure the patient blood drug free fraction, which is considered the active fraction in terms of both drug effect and toxicity. In order to reach the low limit of detection required by the clinicians and enable the detection of the therapeutic drug free fraction, a heterogeneous binding inhibition immunoassay has been developed which makes use of antibody-coated fluorescent and magnetic nanoparticles. The microfluidic optical chip, which exploits total internal reflection fluorescence (TIRF) and fluorescence anisotropy, is constituted by an array of microfluidic channels whose surface is chemically modified with the analyte derivative. The excitation light, coming from an external source, is properly coupled and confined by total internal reflection into the optical waveguide constituting the chip, and is guided towards the sensing area. After binding with the analyte derivative immobilised on the microchannel surface, the fluorescent-magnetic nanoparticles, coated with the analyte-specific antibodies, can be excited by means of the evanescent field, which arises at the waveguide/chip surface, and the emitted fluorescence can be collected by means of large area photodiodes. A thorough study on the excitation and emission of a fluorophore near a dielectric interface was undertaken theoretically and experimentally in order to optimize the microfluidic chip design for the best fluorescence collection efficiency. Different materials have been also investigated and several chip prototypes have been realized and characterized. The final microfluidic optical chip consists of three different polymeric parts bonded together: a thin Zeonor foil (188 µm thick, R.I. = 1.53) which is used as excitation waveguide, a double side adhesive tape (140 µm thick, R.I. = 1.49), in which ten channels are structured by laser cutting and in which the heterogeneous inhibition binding immunoassay is performed, and a Zeonex slide (1 mm thick, R.I = 1.51). The developed microfluidic optical chip is capable of performing the simultaneous analysis of three different selected analytes (Tacrolimus, Cyclosporin A and Mycophenolic acid), each of them measured three times in three different channels with an additional channel for waste flowing. One of the main challenges of the work is to make the microfluidic optical chip easy to use and easy to integrate with different functional elements into a POCT device. These elements include the optoelectronic system (for both the excitation and the detection of the fluorescent signal), the magnetic trapping system (for the attraction and confinement of magnetic and fluorescent nanoparticles within the sensing area), and the fluidic system (for the automatic management of samples and reagents). Specifically, for the optoelectronic excitation system, different laser sources and different optical arrangements for the in-coupling of the light into the excitation waveguide have been experimentally evaluated and tested. An optimized butt-coupling illumination system based on an optical fibre bundle has been finally developed and integrated with the microfluidic chip. As second step of integration, the optical acquisition system, constituted of amorphous large area silicon (a-Si:H) photodiodes, absorption optical filters and readout electronics, has been properly developed in collaboration with the partners of the project. In order to favour the interaction between the fluorescent magnetic nanoparticles and the sensing layer, two magnetic trapping strategies based on current line structures (magnetic coils) and on permanent magnets have been evaluated, and a permanent magnet platform has been properly designed to be finally coupled with the microfluidic optical chip. The automatic management of the fluidics is an essential requirement for the development of POCT devices: from this point of view a fluidic system has been optimized for the implementation of the heterogeneous assay into the channels of the chip. A laboratory version of the integrated system has been developed and it can be considered as a transition setup. The final NANODEM POCT device has been finally manufactured and its functionality has been evaluated.