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Microfluidic cancer cell sorting using dielectrophoresis

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Authors
Balogh, Péter
Date
2020
Discipline
Soveltava fysiikkaApplied Physics
Copyright
This publication is copyrighted. You may download, display and print it for Your own personal use. Commercial use is prohibited.

 
Mikrofluidiikka mahdollistaa mikrobiologisten partikkeleiden, kuten solujen, viruksien, proteiinien ja DNA:n analysoinnin. Se pystyy täyttämään näiden tiukat elinolojen vaatimukset, joita ovat paine, lämpötila ja kemiallinen koostumus. Tarkoituksena oli kehittää valmistusmenetelmä mikrofluidiselle sirulle, joka pystyisi erottelemaan yksittäisiä fluoresenssilla merkattuja syöpäsoluja. Sirussa kulkevia soluja tarkasteltaisiin spektroskopian menetelmin, ja mikäli solu ilmaisisi fluoresenssia, dielektroforeesinen voima ohjaisi sen erilleen talteen otettavaksi. Valmistamalla tällainen siru, joka pystyisi analysoimaan verinäytteestä tuhansia soluja sekunnissa ja erottelemaan niistä yksittäiset syöpäsolut, pystyttäisiin vaikuttamaan merkittävästi syöpätutkimuken kehitykseen. Lasi on optimaalinen valinta tällaiselle sirulle, sillä sen läpinäkyvyys ja kemiallinen vakaus mahdollistvat spektroskopian ja mikrofluidisen ympäristön tarjoamisen. Tästä syystä siru oli kehitetty käyttämällä substraattina soda-lime lasia. Käyttämällä elektronisuihkulitografiaa, fysikaalista kalvonkasvatusmenetelmää sekä muita mikrofabrikaation työkaluja, siru saatiin valmistettua. Dielektroforeesia varten valmistettiin elektrodit, jotka koostuivat Ti, Au ,Ti ja SiO2 kerroksista. Ne olivat tarpeeksi kestäviä selvitääkseen lasien bondaukseen tarvittavasta korkeasta lämpötilasta (585 °C) sekä hyvin syövyttävästä Piranha liuoksesta. Sirun puhdistusmenetelmät, etsausmaski sekä etsausparametrit optimoitiin tasaisen ja virheettömän pinnan aikaansaamiseksi. Etsausmaskina käytettiin 50 nm Cr ja 200 nm + 100 nm Au, joka kesti 30 µm syvien kanavien etsauksen vetyfluoridilla ja pystyi suojelemaan sen alla olevia elektrodeja. Koejärjestely suoritettiin dielektroforeettisen erottelun havainnollistamiseksi fluoresoivilla polystyreeni partikkeleilla. Partikkelit saatiin onnistuneesti virtaamaan kanavien läpi, mutta niiden dielektroforeettinen ohjaaminen epäonnistui. Tähän syynä oli luultavasti viallinen elektrodi. Uuden sirun valmistus ei ollut mahdollista laitoksen ollessa suljettuna covid-19 pandemian vuoksi. Sirun valmistusohjeet ja niiden parantamisehdotukset antavat kuitenkin vahvan perustan projektin jatkamiselle. ...
 
Microfluidics grant the possibility for the analysis of microbiological particles such as cells, viruses, proteins, and DNA. It can fulfil the strict requirements for temperature, pressure, and chemical composition set by samples in vivo environment. The aim was to develop the fabrication of a microfluidic chip that would be capable of single cell sorting, fluorescently labelled cancer cells to be more precise. Cells flowing through the system would be analysed using spectroscopy to detect fluorescence. Depending on the result, a dielectrophoretic force would be exerted on the target cell guiding it to a corresponding channel to be collected or discarded. Achieving a high throughput device that could analyse thousands of cells a second while sorting individual cancer cells from blood samples would have a significant impact on cancer research. Glass, being transparent and chemically inert, is an optimal material for spectroscopic analysis and as a microfluidic environment. Thus, a fabrication recipe for a glass (Soda-lime) microfluidic chip including electrodes was developed. Using electron beam lithography and thin film deposition, such a chip was realised with minimised defects. The electrodes consisted of a sandwich of Ti, Au, Ti and SiO2. They could withstand the highly corrosive Piranha treatment for surface activation and the temperature of 585 °C required for the glass bonding. The cleaning procedure of the chip, etch mask, and etching parameters were optimized to minimize pinhole generation and channel roughness. As an etch mask, thin film layers of 50 nm Cr and 200 nm + 100 nm Au were used. It enabled a near defect free etching of 30 µm deep channels in HF while protecting the underlining electrodes. An experimental setup was assembled to demonstrate deflection of fluorescent polystyrene particles illustrating cells. The proper flow of fluorescent particles was established, but the sorting was unsuccessful most likely due to a faulty electrode. The fabrication of a new chip was unfortunately not possible because of the facility lockdown due to the covid-19 pandemic. However, the fabrication recipe and design improvement ideas presented here grant a good foundation for future continuations on this project. ...
 
Keywords
glass microfluidics wet etching of glass dielectrophoresis DEP cell sorting fabrication mikrofluidistiikka syöpäsolut ohutkalvot solut fysikaalinen kemia microfluidics cancer cells thin films cells physical chemistry
URI

http://urn.fi/URN:NBN:fi:jyu-202007305439

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