Caputo, Tania Mariastella
(2018)
Tuneable hydrogel platform for oligonucleotide biomarkers detection.
[Tesi di dottorato]
Abstract
Early detection of circulating biomarkers in human fluids can improve the quality of life reducing the development of several deadliest diseases. Among the latest and most significant medical concept “Liquid Biopsy” is emerging as non-invasive method of gleaning insight into the dynamics of diseases through a patient fluid sample. Actually many tests have been developed in this context, however, despite all the efforts, the majority are complex, require extensive manipulations and skilled operators, failing for point-of-care (POC) applications.
The main focus of this thesis has been devoted to develop advanced technologies, based on a hydrogel platform, properly designed for biosensing application. In particular, PEG engineered hydrogel microparticles have been synthetized with different chemical strategies and functionalized with oligonucleotide probes to detect circulating biomarker in human serum. In this thesis, the parameters affecting the hydrogel biosensing properties have been carefully evaluated to obtain an accurate functionalized network capable of sensitive and specific biomarker recognition. The developed hydrogel assays are based on the optical fluorescence read out over a single microgel, fixed the number of microgels and sample volume for each test. Therefore, the target concentration is easily quantified comparing the fluorescence observed with a calibration curve.
The thesis starts with the description of a microgel-based bioassay for Cytomegalovirus infection diagnosis (Chapter 2). The bioassay is based on microgels, with core-shell architecture, endowed with optical fluorescence probes for the recognition of circulating endogenous viral hcmv-miR-US4-5p. Then, a microgel-based bioassay for microRNA biomarker detection in cancer application is reported (Chapter 3). In this case, the outmost shell of microgel is functionalized with molecular beacons for circulating miR-21 recognition. In Chapter 4 are elucidated the main parameters taken into consideration to develop sensitive and specific microgel-based assay for long oligonucleotide detection, as lncRNA, mRNA or DNA. In particular, core-shell microgels functionalized with double strand or molecular beacon probes are compared in terms of sensitivity, sensibility and assay time. Finally, is described the design of Three-dimensional hydrogel microparticles in microfluidics for the detection of microRNA and in particular is presented the case study of miR-143-3p detection as early biomarker in Amyotrophic lateral sclerosis (Chapter 5).
Results show that finely tuning both the probe density and the number of microparticles per assay are achieved appealing limits of detection, avoiding amplification steps. The molecular beacon-microgels assay further reduces the characteristics time of hybridization observed in beads based assay and is extremely specific towards single mutated targets (SNP). Due to the PEG anti-fouling property, target detection occurs in human serum without loss in sensitivity. Moreover, the hydrogel-based assays are suitable for several laboratory equipment, are stable over a one-year span time and work in low sample volume. In addition to the mentioned advantages, microfluidics approach significantly reduces costs and the time of production resulting attractive for industrial production.
Therefore, the biosensing platform obtained using engineered hydrogels can represent a smart technology capable to predict, identify and follow-up several diseases, monitoring free circulating oligonucleotides in body fluids. The flexible use of these engineered hydrogels, which avoid sample manipulation and can be easily integrated into miniaturized device for optical readout, aims to push these technologies as point-of-care device.
Downloads per month over past year
Actions (login required)
 |
Modifica documento |
