Desenvolupament d'una plataforma lab-on-a-chip per a la realització automatitzada d'assajos d'afinitat amb marca enzimàtica
Recently, having access to tools to diagnose a disease in a rapid way has become of great importance, especially after the pandemic caused by SARS-CoV-2 virus. Currently, the vast majority of analysis are carried out by technicians in specialized labs. In this way, the span time between the sampling...
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| Format: | doctoral thesis |
| Status: | Versión aceptada para publicación |
| Publication Date: | 2022 |
| Country: | España |
| Institution: | Consejo Superior de Investigaciones Científicas (CSIC) |
| Repository: | DIGITAL.CSIC. Repositorio Institucional del CSIC |
| OAI Identifier: | oai:digital.csic.es:10261/340428 |
| Online Access: | http://hdl.handle.net/10261/340428 |
| Access Level: | Open access |
| Keyword: | Lab-On-A-Chip Elisa Vàlvules microfluídiques Válvulas microfluídicas Microfluidic valves |
| Summary: | Recently, having access to tools to diagnose a disease in a rapid way has become of great importance, especially after the pandemic caused by SARS-CoV-2 virus. Currently, the vast majority of analysis are carried out by technicians in specialized labs. In this way, the span time between the sampling and the results is at least 48h. This delay hinders the possibility to act rapidly and accordingly to the results obtained. Therefore, having fast and robust analysis tools that could be used at the same point-of-care is a considerable improvement in the diagnosis and monitoring of a disease. Some of these tools are already available in the market, like for example the pregnancy test, the test for glucose detection for diabetic patients or the SARS-CoV-2 test. However, only simple analysis, that require few reagents and steps, can be done with these devices. Moreover, the obtained result is often non-quantitative, which limits the use of these devices to the applications where no quantification is required. The development of lab-on-a-chip devices to overcome these limitations requires to miniaturize and incorporate other elements, like more precise detection systems or fluid handling systems (e.g. valves and pumps). Nevertheless, the majority of these devices rely on bulky elements that restrict its use in-situ. This thesis is based on the wax valve technology previously developed by the Grup de Transductors Químics of Institut de Microelectrònica de Barcelona (IMB-CNM, CSIC). The first valves were electrically actuated, but finally they were adapted to be controlled by LED light pulses which enables to simplify the interface and develop a potentially more compact platform. Taking these valves as the starting point, the thesis is focused on adapting the technology to integrate them into microfluidic chips fabricated by rapid prototyping techniques. In addition, taking advantage of the benefits of these valves, a portable instrument to automate the use of the chips, that incorporates a pumping system and an absorbance measurement system, has been developed. A part from that, the platform has been adapted to develop ELISA-like (Enzyme-Linked Immunosorbent Assay) affinity assays with enzymatic labelling. To perform those assays, a solid phase, in which the capture molecules have been immobilized, is incorporated inside the chip. The fluidic behaviour of three different solid phases has been studied: polystyrene microspheres, nitrocellulose and glass. Later, an ELISA sandwich like assay (onto nitrocellulose) and an ELONA (Enzyme-Linked Oligonucleotide Assay) (onto glass) have been implemented on-chip. To optimize the assays, chips without valves and a commercial syringe pump have been used to guarantee the ideal control of fluidic parameters. The first assay is based on the detection of tumour necrosis factor alpha (TNFα), a pro-inflammatory protein widely used as a biomarker to study the anti-inflammatory response to various diseases. The second assay is based on the detection of 16S rRNA sequence of Escherichia coli by the interaction between this sequence with complementary ones in an ELONA like assay. E.coli is one of the most common bacteria causing infections, both in humans and animals. That is why it is a good indicator of interest in areas like medicine, the food industry or the environment. The viability to perform automatic immunoassays has been demonstrated although some needs for design improvements have been detected which should be addressed in order to maximize the potential of the developed platform. |
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