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.

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.

Effective isolation and purification of circulating tumor cells from whole blood provides important capability for clinical application and biological research. Here, we demonstrate a single step surface modification procedure for a microfluidic device based on self-assembly of recombinant spider silk harbouring an affinity domain for antibody binding. Moreover, a proteolytic domain for enzymatic digestion of silk for effective release of cancer cells from whole blood of Pancreatic patient sample and the released cells are readily available for downstream processing.

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.

Layer-by-layer (LBL) technique facilitates the production of the thin coating of cellulose onto polymeric surfaces and modified to form affinity based cell capture surface. We demonstrate an efficiently capture and release of cells, the release is done by selectively degrading the cellulose layers using enzyme and cells can be collected without losing cell viability.

According to reports by the World Health Organization (WHO), cancer-related deaths reached almost 10 million in 2018. Nearly 65% of these deaths occurred in low- to middle-income countries, a trend that is bound to increase since cancer diagnostics are not currently considered a priority in resource-limited settings (RLS). Thus, cost-effective and specific cancer screening and diagnostics tools are in high demand, particularly in RLS. The selective isolation and up-concentration of rare cells while maintaining cell viability and preventing phenotypic changes is a powerful tool to allow accurate and sensitive downstream analysis. Here, multi-layer cellulose nanofibril-based coatings functionalized with anti-EpCAM antibodies on the surface of disposable microfluidic devices were optimized for specific capture of target cells, followed by efficient release without significant adverse effects. HCT 116 colon cancer cells were captured in a single step with >97% efficiency at 41.25 mu L min(-1) and, when spiked in whole blood, an average enrichment factor of similar to 200-fold relative to white blood cells was achieved. The release of cells was performed by enzymatic digestion of the cellulose nanofibrils which had a negligible impact on cell viability. In particular, >80% of the cells were recovered with at least 97% viability in less than 30 min. Such performance paves the way to expand and improve clinical diagnostic applications by simplifying the isolation of circulating tumor cells (CTCs) and other rare cells directly from whole blood.

The Basophil Activation Test (BAT) is a valuable allergy diagnostic tool but is time-consuming and requires skilled personnel, which has limited its clinical use. We therefore developed and clinically tested a microfluidic immunoaffinity BAT (miBAT) technique where we captured basophils directly from whole blood followed by in vitro activation and quantification of activation markers. For the first time basophils captured from whole blood, from both allergic patients and healthy donors, have been activated using allergens.

Background: The flow cytometry-based basophil activation test (BAT) is used for the diagnosis of allergic response. However, flow cytometry is time-consuming, requiring skilled personnel and cumbersome processing, which has limited its use in the clinic. Here, we introduce a novel microfluidic-based immunoaffinity BAT (miBAT) method. Methods: The microfluidic device, coated with anti-CD203c, was designed to capture basophils directly from whole blood. The captured basophils are activated by anti-FceRI antibody followed by optical detection of CD63 expression (degranulation marker). The device was first characterized using a basophil cell line followed by whole blood experiments. Weevaluated the device with ex vivo stimulation of basophils in whole blood from healthy controls and patients with allergies and compared it with flow cytometry. Results: The microfluidic device was capable of capturing basophils directly from whole blood followed by in vitro activation and quantification of CD63 expression. CD63 expression was significantly higher (P = 0.0002) in on-chip activated basophils compared with nonactivated cells. The difference in CD63 expression on anti-FceRI-activated captured basophils in microfluidic chip was significantly higher (P = 0.03) in patients with allergies compared with healthy controls, and the results were comparable with flow cytometry analysis (P = 0.04). Furthermore, there was no significant difference of CD63% expression in anti-FceRI-activated captured basophils in microfluidic chip compared with flow cytometry. Conclusions: We report on the miBAT. This device is capable of isolating basophils directly from whole blood for on-chip activation and detection. The new miBAT method awaits validation in larger patient populations to assess performance in diagnosis and monitoring of patients with allergies at the point of care.

Mass spectrometry-based proteomics benefits from efficient digestion of protein samples. In this study, trypsin was immobilized on nanoporous anodized alumina membranes to create an enzyme reactor suitable for peptide mass fingerprinting. The membranes were derivatized with 3-aminopropyltriethoxysilane and the amino groups were activated with carbonyldiimidazole to allow coupling of porcine trypsin via c-amino groups. The function was assessed using the artificial substrate Na-Benzoyl-L-arginine 4-nitroanilide hydrochloride, bovine ribonuclease A and a human plasma sample. A 10-membrane flow-through reactor was used for fragmentation and MS analysis after a single pass of substrate both by collection of product and subsequent off-line analysis, and by coupling on-line to the instrument. The peptide pattem allowed correct identification of the single target protein in both cases, and of > 70 plasma proteins in single pass mode followed by LC-MS analysis. The reactor retained 76% of the initial activity after 14 days of storage and repeated use at room temperature. Significance: This manuscript describes the design of a stable enzyme reactor that allows efficient and fast digestion with negligible leakage of enzyme and enzyme fragments. The high stability facilitates the use in an online-setup with MS detection since it allows the processing of multiple samples within an extended period of time without replacement.

Background: Bloodstream infections (BSI) remain a major challenge with high mortality rate, with an incidence that is increasing worldwide. Early treatment with appropriate therapy can reduce BSI-related morbidity and mortality. However, despite recent progress in molecular based assays, complex sample preparation steps have become critical roadblock for a greater expansion of molecular assays. Here, we report a size based, label-free, bacteria separation from whole blood using elasto-inertial microfluidics.

Results: In elasto-inertial microfluidics, the viscoelastic flow enables size based migration of blood cells into a non- Newtonian solution, while smaller bacteria remain in the streamline of the blood sample entrance and can be sepa- rated. We first optimized the flow conditions using particles, and show continuous separation of 5 μm particles from 2 μm at a yield of 95% for 5 μm particle and 93% for 2 μm particles at respective outlets. Next, bacteria were continu- ously separated at an efficiency of 76% from undiluted whole blood sample.

Conclusion: We demonstrate separation of bacteria from undiluted while blood using elasto-inertial microfluidics. The label-free, passive bacteria preparation method has a great potential for downstream phenotypic and molecular analysis of bacteria.