Titolo della tesi: LAB ON CHIP SYSTEM FOR THE DETECTION OF PATHOGENS AND METABOLITES IN BIOLOGICAL SAMPLES
Lab-on-Chip (LoC) is a miniaturized device that integrates on a single substrate one or multiple analyses, which are usually done in a laboratory. LoC may include liquid handling, temperature control, and sample detection in a miniaturized and compact system. LoCs have been demonstrated to lead to faster analysis and response times due to short diffusion distances, fast heating, high surface-to-volume ratios, small heat capacities, and massive parallelization due to compactness, which allows high-throughput analysis. Most LOCs are novel proof-of-concept applications that are not yet fully developed for widespread use.
In this thesis, the focus is to investigate the possibility of performing biochemical analysis for diagnostic purposes using two types of developed LoCs. In particular, the detection of pathogens and different metabolites in biological samples is displayed:
1. detection of viruses such as watermelon mosaic virus (WMV) and SARS Cov-2 using an LoC to perform reverse-transcriptase real-time PCR (RT-qPCR);
2. detection of different metabolites in biological samples using an aptamer assay integrated with an LoC for fluorescent detection, indicating the presence of a pathogen or the metabolite variation in a biological sample.
The LoC systems used for this work are based on microfluidic for sample handling coupled with an optoelectronic device (OD). The OD allows temperature generation through the presence of thin-film heaters, which generate the heat, and temperature sensors and photosensors, based on amorphous silicon, to control the temperature and detect the fluorescent light deriving by the detected target, respectively.
The direct detection of viruses is demonstrated using the on-Chip RT-qPCR for two types of viruses, one plant pathogenic virus named WMV, and a human virus, the Sars-CoV-2. In this system, the OD includes thin-film heaters, temperature sensors, and photosensors. The OD is coupled with a properly designed glass-PDMS (polydimethylsiloxane) microwell plate. The RNA extracted from the virus is analyzed in 10-fold dilution samples showing an analytical sensitivity down to 10-6, demonstrating the LoC system can be used as a miniaturized portable device for performing RT-qPCR for the detection of the virus.
The use of an LoC system for detecting metabolites is demonstrated by using different assays for the detection of ATP and Zinc (ZnCl2). In these systems, the assay is based on the use of aptamer sequences.
Aptamers are short sequences of oligonucleotides that bind to specific target molecules. Aptamers are generated by in vitro selection process (SELEX) with a high degree of purity and high reproducibility. Aptamers are chosen over antibodies due to their low-cost production, stability, specificity, and high target potential.
The LoC system for the detection of ATP and Zinc consists of an OD base on an array of photosensors which are optically coupled with a microfluidic device including ten channels made of pressure adhesive tape (PSA) bonded to a glass substrate. The microfluidic allows the simultaneous detection of ten samples and is designed to permit the sample to flow by capillary force without the need for an external pumping system. The microfluidic channels are functionalized with a layer of poly (2-hydroxyethyl methacrylate) polymer brushes (PHEMA) to which the aptamer sequence is covalently bonded. Solutions containing different concentrations of metabolites flow in the functionalized microfluidic device. Upon interaction with the aptamers, those undergo a change of conformation and in presence of a fluorescent intercalating dye, there is a modification of the fluorescent intensity, which is detected by the photosensors positioned underneath. The LoC showed the detection down to a concentration of about 1 µM, in standard solutions and down to 10 CFU/mL in bacterial solutions.
Solutions of Zinc with a concentration down to 1 µM are detected using the LoC system, displaying a higher sensitivity compared with that obtained using the spectrofluorometric analysis.