Aliberti, Anna (2013) Multiplexed Microgels For Oligonucleotides Detection. [Tesi di dottorato]

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Abstract

The development of rapid detection strategies toward point-of-care applications has been receiving increasing attention due to the time and labor intensive protocols associated with well-established assays. In particular, assays that are free of separation, amplification, and other related operations are highly desirable. In recent years, the use of encoded beads has received considerable attention due to their potential for measuring multiple analytes in solution. This can be achieved without the need for knowledge of their spatial position, as in the case of microarray technology. Encoded bead technology also relies on the solution kinetics rather than diffusion to a fixed surface as in the case of microarray technology, offering the possibility of developing rapid high throughput screening methods. Attempts to develop this technology tend to focus on the generation of featured barcodes both with a large number of identifications to increase the throughput and with novel functions to improve the assays. This thesis describes a synthesis, characterization and unique properties of multi-responsive encoded core shell microgel. for the direct detection in multiplex of single strands nucleic acids (ssDNA, miRNA etc) at very low concentration (femtomolar), without the need of other conventional tools such as PCR, Southern blot or microarray. The encoded microgels are provided by a wide range of fluorescence-based codes with an innovative core-shell material architecture. However, compared to the already validated bead based detection systems, microgel composed by bio inert PEG are no fouling with minimal nonspecific binding that makes them favorable for assays in complex biological samples. The platform assay, named MEDiA (Microgel Enhanced Displacement Assay) consists of innovative probes, that mounted on PEG-encoded microgels, are able to capture and reveal the presence of the complementary oligonucleotides strand of DNA or miRNA through fluorescence emission. The conjugation on particle surface brings a large number of fluorofore probes into a small region which significantly increases the fluorescence intensity and facilitates further manipulation. Here we provide the proof of concept of the assay by using genetic material specific for viruses such as SARS, HIV, HCV and RNA, i.e. miRNAs. The detection mechanism is based on a double strand displacement assay. Compared to other homogeneous assays for nucleic acids, ds displacement assay acts as integrated sensor that can i) be mounted on bead surfaces, ii) capture the target molecules and iii) highlight the binding event. The evaluation of nucleotide concentration as well as the code is a result of fluorescence emission analysis over a fixed number of microgels. The flexibility of the proposed platform could allow performing point of care analysis, both where the sensitivity is not a stringent requirement and both where an ultra-sensitive quantification is necessary by using fluorescent microscopy or miniaturized systems (lab-on-chip).

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