An all-fiber integrated device capable of separating and counting particles is presented. A sequence of silica fiber capillaries with various diameters and longitudinal cavities are used to fabricate the component for size-based elasto-inertial passive separation of particles followed by detection in an uninterrupted continuous flow. Experimentally, fluorescent particles of 1 μm and 10 μm sizes are mixed in a visco-elastic fluid and fed into the all-fiber separation component. The particles are sheathed by an elasticity enhancer (PEO - polyethylene oxide) to the side walls. Larger 10 μm particles migrate to the center of the silica capillary due to the combined inertial lift force and elastic force, while the smaller 1 μm particles are unaffected, and exit from a side capillary. A separation efficiency of 100% for the 10 μm and 97% for the 1 μm particles is achieved at a total flow rate of 50 μL min−1. To the best of our knowledge, this is the first time effective inertial-based separation has been demonstrated in circular cross-section microchannels. In the following step, the separated 10 μm particles are routed through another all-fiber component for counting and a counting throughput of ∼1400 particles per min is demonstrated. We anticipate the ability to combine high throughput separation and precise 3D control of particle position for ease of counting will aid in the development of advanced microflow cytometers capable of particle separation and quantification for various biomedical applications.

Three-dimensional multicellular spheroids (MCSs) are complex structure of cellular aggregates and cell-to-matrix interaction that emulates the in-vivo microenvironment. This research field has grown to develop and improve spheroid generation techniques. Here, we present a new platform for spheroid generation using Layer-by-Layer (LbL) technology. Layer-by-Layer (LbL) containing cellulose nanofibrils (CNF) assemble on a standard 96 well plate. Various bi-layer numbers, multiple cell seeding concentration, and two tumor cell lines (HEK 293 T, HCT 116) are utilized to generate and characterize spheroids. The number and proliferation of generated spheroids, the viability, and the response to the anti-cancer drug are examined. The spheroids are formed and proliferated on the LbL-CNF coated wells with no significant difference in connection to the number of LbL-CNF bi-layers; however, the number of formed spheroids correlates positively with the cell seeding concentration (122 ± 17) and (42 ± 8) for HCT 116 and HEK 293T respectively at 700 cells ml−1. The spheroids proliferate progressively up to (309, 663) µm of HCT 116 and HEK 293T respectively on 5 bi-layers coated wells with maintaining viability. The (HCT 116) spheroids react to the anti-cancer drug. We demonstrate a new (LbL-CNF) coating strategy for spheroids generation, with high performance and efficiency to test anti-cancer drugs.

Efficient separation and detection of rare cells in a mixed population is important in many biomedical applications. For instance, isolating and detecting circulating tumor cells (CTCs) from whole blood samples could allow for early cancer diagnosis and prognosis during treatment. CTCs are rare cells circulating in blood detached from the primary tumor site, carrying important information such as the origin of cancer and metastatic information. The detection of CTC from blood samples, besides being a minimally invasive procedure, could be vital in case of difficulty to access the tumor site via traditional biopsies, such as colon and pancreatic cancer. Microfluidics is a research field with great promise towards the development of methods to isolate and separate cells for clinical applications. Microfluidic based cell separation has been demonstrated using biological approaches using cell surface markers, and biophysical approaches using cell size, shape, and deformability. This thesis will focus on developing passive strategy using inertial microfluidics (biophysical, paper 1-4) and affinity biomarker (biochemical, paper 5) based strategy to isolate and analyze CTCs. Inertial microfluidics relies on inherent hydrodynamic forces, inertial forces, in flow through the microfluidic channel. Depending on the geometry of the channel, inertial forces drive the particles and cells to a specific streamline position, allowing for focusing and separation. In contrast, affinity-based isolation relies on biomarkers expressed on the surface of the targeted cells, which is highly specific. In paper 1, using the elasto inertial microfluidic technique, high throughput particle focusing and separation was achieved in a curved rectangular channel with a separation efficiency of 89% for 10 μm and 99% for the 15 μm particles at a high volumetric flow rate (1 mL/min). In paper 2, a detailed analysis of particle focusing was studied experimentally and numerically in a circular cross-section. Using the FENE-P model simulating non-Newtonian fluid and an immersed boundary method to account for the particles, it was observed that a combination of inertia and elasticity leads to several intermediate focusing positions. In paper 3, we developed a portable microflow cytometer using fiberoptics capillaries. By combining elasto inertial microfluidics and optical fibers, we focused particles and cells and demonstrated particle counting at a throughput of 2500 particles/second. In paper 4, we built an all-fiber separation and detection component and demonstrated a separation efficiency of 100% for the 10 μm and 97% for the 1 μm particles as a proof of principle. In addition, the separated 10 μm particles could beiiiquantified in the all-fiber component. In paper 5, an affinity-based separation approach was carried out to utilize the surface markers to capture and release viable CTCs for downstream analysis. A novel layer-by-layer nanofilm coating strategy was developed using cellulose nanofibril (CNF) built into multiple layers and functionalized with antibodies to capture the cells. After capture, the CNF were enzymatically degraded to release the CTCs. HCT116 colon cancer cells were captured with an efficiency of more than 97%, and when spiked in whole blood, an approximately 200 fold average enrichment was achieved compared to white blood cells. 80% of the cancer cells spiked in whole blood were recovered with 97% viability in less than 30 minutes.

In summary, this thesis presents different microfluidics-based separation of cancer cells based on biophysical and biochemical properties. Using elasto inertial microfluidics, we developed several approaches to separate and detect cells and particles. Using layer-by-layer coating of CNF, we successfully demonstrated capture and release of cancer cells with maintained high viability. While the thesis has focused on different properties of cells for separation and analysis, combining these methods will be important for efficient isolation and characterization of CTCs for improved diagnostics.

Separationen och detektionen av specifika celler i en blandad population av celler är viktig i många biomedicinska tillämpningar. Som exempel, möjligheten att isolera och detektera cirkulerande tumörceller (CTC) från helblod skulle kunna tillåta tidig cancer diagnos och prognos under behandling. CTC är sällsynta celler som cirkulerar i blodet och bär med sig viktig information, som den specifika cancerns ursprung och metastatiska information. Att kunna detektera CTC med hjälp av blodprover, förutom att erbjuda en minimalt invasiv metod, skulle kunna vara viktig i fall där tumörområdet är svårtillgänglig för traditionell provtagning via biopsier, såsom kolon- och bukspottkörtel-cancer.

Mikrofluidik är ett forskningsfält med betydande potential att möjliggöra utvecklingen av metoder för att isolera och separera celler för kliniska tillämpningar. Separation av celler baserad på mikrofluidik har demonstrerats med olika angreppssätt så som biologiska med hjälp av affinitetsmarkörer, och biofysiska metoder där man utnyttjar storlek, form, och deformerbarhet för att separera celler. Denna avhandling fokuserar på att utveckla en passiv strategi som utnyttjar tröghets-baserade mikrofluidik som domineras av tröghetskrafter (papper 1-4) och strategier med affinitetsbiomarkörer (paper 5) med målet att isolera och analysera CTC. Tröghetsfokusering i mikroflöden baseras på hydrodynamiska krafter, tröghetskrafter, som utvecklas i vätskeflöden i mikrokanaler. Beroende på mikrokanalens geometriska utformning och vätskans flödes hastighet kommer tröghetskrafterna att driva partiklar eller celler till specifika positioner i strömningsfältet och i sin tur möjliggöra fokusering och separation. Å andra sidan, affinitetsbaserad isolering är beroende på biomarkörer som uttrycks på ytan av specifika celler och är därmed mycket specifik. I papper 1 utnyttjas mikrofluidisk metod med tröghetskrafter med elastiska bidrag för att möjliggöra partikel fokusering och separation vid höga volymsflöden. I papper 2, en detaljerad analys av partikelfokusering i en circulärt tvärsnitt genomfördes experimentellt och numeriskt. I papper 3, en portabel mikroflödescytometer utvecklades med hjälp av fiberoptiska kapillärer. Med hjälp av mikrofluidik som utnyttjar elastiska och tröghetskrafter tillsammans med optiska fibrer, fokuserades partiklar och celler och demonstrerade möjligheten att räkna partiklar och celler. I papper 4 beskrivs en fiber-baserad komponent för separation och detektion som demonstrerade en separationseffektivitet av 100% för 10 µm-partiklar och 97% för 1 µm-partiklar som ett bevis på principen. I papper 5, en affinitetsbaserad separationsmetod utvecklades för att utnyttja ytmarkörer som finns på cirkulerande tumörceller. En beläggningsstrategi med hjälp av nanocellulosa utvecklades för att först fånga in och och sedan frigöra levande CTC för vidare analys. Den nya nanocellulosa-baserade ytbeläggningen fångar och frigör celler med hjälp av en enzym för analys nedströms. Sammanfattningsvis, denna avhandling presenterar mikrofluidik-baserad separation av cancerceller som utnyttjar biofysiska och biokemiska egenskaper. Med hjälp av tröghetsfokusering i mikrofluidik utvecklades flera metoder för att separera och detektera celler och partiklar. Dessutom utvecklades en original metod som bygger på att ytbehandla chip med nanocellulosa för infångning och frigörande av CTCs. I avhandlingen har vi undersökt olika metoder för isolering and analys av cancer celler. Medan varje metod har sin fördel och svaga punkter, kommer det att vara viktigt att kombinera dessa metoder och andra för att bidra till bätter cancer diagnostik i framtiden.

We report a unique tuneable analogue trend in particle focusing in the laminar and weak viscoelastic regime of elasto-inertial flows. We observe experimentally that particles in circular cross-section microchannels can be tuned to any focusing bandwidths that lie between the “Segre-Silberberg annulus” and the centre of a circular microcapillary. We use direct numerical simulations to investigate this phenomenon and to understand how minute amounts of elasticity affect the focussing of particles at increasing flow rates. An Immersed Boundary Method is used to account for the presence of the particles and a FENE-P model is used to simulate the presence of polymers in a Non-Newtonian fluid. The numerical simulations study the dynamics and stability of finite size particles and are further used to analyse the particle behaviour at Reynolds numbers higher than what is allowed by the experimental setup. In particular, we are able to report the entire migration trajectories of the particles as they reach their final focussing positions and extend our predictions to other geometries such as the square cross section. We believe complex effects originate due to a combination of inertia and elasticity in the weakly viscoelastic regime, where neither inertia nor elasticity are able to mask each other’s effect completely, leading to a number of intermediate focusing positions. The present study provides a fundamental new understanding of particle focusing in weakly elastic and strongly inertial flows, whose findings can be exploited for potentially multiple microfluidics-based biological sorting applications. 

Improved sample preparation has the potential to address unmet needs for fast turnaroundsepsis tests. In this work, we report elasto-inertial based rapid bacteria separation from diluted blood at high separation efficiency. In viscoelastic flows, we demonstrate novel findings where blood cells prepositioned at the outer wall entering a spiral device remain fullyfocused throughout the channel length while smaller bacteria migrate to the opposite wall.Initially, using microparticles, we show that particles above a certain size cut-off remainfully focused at the outer wall while smaller particles differentially migrate toward the inner wall. We demonstrate particle separation at 1 μm resolution at a total throughput of1 mL/min. For blood-based experiments, a minimum of 1:2 dilution was necessary to fullyfocus blood cells at the outer wall. Finally, Escherichia coli spiked in diluted blood were continuously separated at a total flow rate of 1 mL/min, with efficiencies between 82 and 90%depending on the blood dilution. Using a single spiral, it takes 40 min to process 1 mLof blood at a separation efficiency of 82%. The label-free, passive, and rapid bacteria isolation method has a great potential for speeding up downstream phenotypic and genotypicanalysis.

Passive particle manipulation using inertial and elasto-inertial microfluidics have received substantial interest in recent years and have found various applications in high throughput particle sorting and separation. For separation applications, elasto-inertial microfluidics has thus far been applied at substantial lower flow rates as compared to inertial microfluidics. In this work, we explore viscoelastic particle focusing and separation in spiral channels at two orders of magnitude higher Reynolds numbers than previously reported. We show that the balance between dominant inertial lift force, dean drag force and elastic force enables stable 3D particle focusing at dynamically high Reynolds numbers. Using a two-turn spiral, we show that particles, initially pinched towards the inner wall using an elasticity enhancer, PEO (polyethylene oxide), as sheath migrate towards the outer wall strictly based on size and can be effectively separated with high precision. As a proof of principle for high resolution particle separation, 15 mu m particles were effectively separated from 10 mu m particles. A separation efficiency of 98% for the 10 mu m and 97% for the 15 mu m particles was achieved. Furthermore, we demonstrate sheath-less, high throughput, separation using a novel integrated two-spiral device and achieved a separation efficiency of 89% for the 10 mu m and 99% for the 15 mu m particles at a sample flow rate of 1 mL/min-a throughput previously only reported for inertial microfluidics. We anticipate the ability to precisely control particles in 3D at extremely high flow rates will open up several applications, including the development of ultra-high throughput microflow cytometers and high-resolution separation of rare cells for point of care diagnostics.

We describe a prototype of an all-fiber microfluidic cytometer capable of analyzing particles ranging from 2-20 µm in diameter. The portable all-fiber cytometer is fabricated using different diameter silica capillaries for flow of microparticles and optical fibers to deliver and collect light. As a proof of concept, we analyze mixed population of beads of three different sizes and classify them with our all-fiber cytometer.

An all-fiber component capable of sorting and counting microparticles based on size is presented. A sequence of silica fiber capillaries were used to fabricate the component for separation and detection. The portable, lab-scale “all-fiber” device was fabricated by assembling different silica fiber capillaries and optical fibers in a Vytran glass processing station. We report elasto-inertial microfluidics based particle migration and focusing and demonstrate high separation efficiency between 10 µm (100%) and 1 µm (97%) microparticles. The separated 10 µm particles were further analyzed for counting in the integrated fiberoptics component at a speed of ~1400 particles/min.

n all-fiber separation component capable of sorting and counting micron-sized particles based on size is presented. A sequence of silica fiber capillaries with various diameters and longitudinal cavities were used to fabricate the component for separation and detection in an uninterrupted flow. Fluorescence microparticles of 1 μm and 10 μm sizes are mixed in a visco-elastic fluid and infused into the all-fiber separation component. Elasto-inertial forces focus the larger particle to the center of the silica capillary, while the smaller microparticles exit from a side capillary. Analysis of the separated particles at the output showed a separation efficiency of 100% for the 10 μm and 97% for the 1 μm particles. In addition, the counting of the larger particles is demonstrated in the same flow. The separated 10 μm particles are further routed through another all-fiber component for counting. A counting speed of ~1400 particles/min and with the variation in amplitude of 10% is achieved. A combination of separation and counting can be powerful tool may find several applications in biology and medicine, such as separation and analysis of exosomes, bacteria, and blood cell sub-populations.

Manipulation of particles and cells in viscoelastic fluids has received substantial interest because this phenomenon provides high-quality focusing. Here we present an enhanced particle focusing and separation in spiral channels, at a ten-fold increase of Reynolds number as compared to current state of the art elasto-inertial microfluidics and report stable particle focusing in spiral low aspect ratio channels at flow rates two magnitudes higher than that previously reported at a high throughput of 2 mL/min is demonstrated with an separation efficiency of 99% for the 15-micron and 91% for the 10-micron particles is demonstrated.