Publicação
Detection of fluorescence by miniaturized in-chip- incorporated microscope for digital Polymerase Chain Reaction (dPCR)
| Resumo: | Classic biopsies are largely used for cancer diagnosis, which are often an invasive procedure. Alternatively, circulating tumour DNA in the blood can be used as liquid biopsy. For DNA amplification, digital polymer- ase chain reaction (dPCR) is becoming the selected procedure for extremely sensitive quantification of nu- cleic acid molecules. Droplet-based microfluidic platforms are widely used in dPCR, since microfluidics allows the efficient partitioning of samples in nanolitre-sized droplets, using oil-water interfaces, which sta- tistically results in one or zero DNA strand target molecule per droplet prior to amplification, showing a positive or negative fluorescent signal during amplification. In the present thesis, it is proposed a novel dPCR detection technique using a miniaturized fluorescence microscope, UCLA Miniscope, integrated in a droplet generator microfluidic device. The Miniscope was developed for neuroscience research due to its portability and lightweight, to be carried by small animals. This fluorescence detection technique is straight- forward, miniaturized, and inexpensive. To assess fluorescence detection sensitivity by the Miniscope, sev- eral fluorescein isothiocyanate (FITC) solutions were tested, with concentrations ranging from 1 nM to 100 μM, and the lowest concentration to present a detectable fluorescence intensity was 1 μM. Moreover, posi- tive and non-template-control PCR mixes of the cancer biomarker c-Myc after end-point amplification con- taining fluorescent DNA-binding dye EvaGreen were tested, and it was possible to discriminate the positive droplets and the non-template-control droplets. This research opens new prospects for the advancement of improved lab-on-a-chip methods designed for dPCR analysis and for further biological and medical applica- tions. |
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| Autores principais: | Barros, Ana Sofia Magro Saldanha |
| Assunto: | Fluorescence detection Microfluidics Lab-on-chip Digital PCR Miniscope |
| Ano: | 2022 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade Nova de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório Institucional da UNL |
| Resumo: | Classic biopsies are largely used for cancer diagnosis, which are often an invasive procedure. Alternatively, circulating tumour DNA in the blood can be used as liquid biopsy. For DNA amplification, digital polymer- ase chain reaction (dPCR) is becoming the selected procedure for extremely sensitive quantification of nu- cleic acid molecules. Droplet-based microfluidic platforms are widely used in dPCR, since microfluidics allows the efficient partitioning of samples in nanolitre-sized droplets, using oil-water interfaces, which sta- tistically results in one or zero DNA strand target molecule per droplet prior to amplification, showing a positive or negative fluorescent signal during amplification. In the present thesis, it is proposed a novel dPCR detection technique using a miniaturized fluorescence microscope, UCLA Miniscope, integrated in a droplet generator microfluidic device. The Miniscope was developed for neuroscience research due to its portability and lightweight, to be carried by small animals. This fluorescence detection technique is straight- forward, miniaturized, and inexpensive. To assess fluorescence detection sensitivity by the Miniscope, sev- eral fluorescein isothiocyanate (FITC) solutions were tested, with concentrations ranging from 1 nM to 100 μM, and the lowest concentration to present a detectable fluorescence intensity was 1 μM. Moreover, posi- tive and non-template-control PCR mixes of the cancer biomarker c-Myc after end-point amplification con- taining fluorescent DNA-binding dye EvaGreen were tested, and it was possible to discriminate the positive droplets and the non-template-control droplets. This research opens new prospects for the advancement of improved lab-on-a-chip methods designed for dPCR analysis and for further biological and medical applica- tions. |
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