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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. Funding details: European Research Council, ERC, ERC- 2013-CoG-616186; Funding details: Vetenskapsrådet, VR, VR 2014-5001; Funding text 1: LB was supported by the European Research Council Grant No. ERC- 2013-CoG-616186, TRITOS, and by the Swedish Research Council (Grant No. VR 2014-5001). The authors acknowledge computer time provided by SNIC (Swedish National Infrastructure for Computing). QC 20220207

Monitoring technologies for Process Analytical Technology (PAT) in mammalian cell cultures are often focusing on the same hand full parameters although a deeper knowledge and control of a larger panel of culture components would highly benefit process optimization, control and robustness. This short review highlights key advances in microfluidic affinity assays and microchip capillary electrophoresis (MCE). Aiming at the miniaturization and integration of PAT, these can detect at-line a variety of metabolites, proteins and Critical Quality Attributes (CQA’s) in a bioprocess. Furthermore, discrete analytical components, which can potentially support the translation of increasingly mature microfluidic technologies towards this novel application, are also presented as a comprehensive toolbox ranging from sample preparation to signal acquisition.

The biopharmaceutical market has been rapidly growing in recent years, creating a highly competitive arena where R&D is critical to strike a balance between clinical safety and profitability. Toward process optimization, the recent development and adoption of new process analytical technologies (PAT) highlight the dynamic complexity of mammalian/human cell culture processes, as well as the importance of fine-tuning and modeling key metabolites and proteins. In this context, simple, rapid, and cost-effective devices allowing routine at-line monitoring of specific proteins during process development and production are currently lacking. Here, we report the development of a versatile microfluidic protein analysis cartridge allowing the multiplexed bead-based immunodetection of specific proteins directly from complex mixtures with minimal hands-on time. Colorimetric quantification of Chinese hamster ovary (CHO) host cell proteins as key impurities, monoclonal antibodies as target biopharmaceuticals, and lactate dehydrogenase as a marker of cell viability was achieved with limits of detection in the 1-10 ng/mL range and analysis times as short as 30 min. The device was further demonstrated for the monitoring of a Rituximab-producing CHO cell bioreactor over the course of 8 days, providing comparable recoveries to standard enzyme-linked immunosorbent assay (ELISA) kits. The high sensitivity combined with robustness to matrix interference highlights the potential of the device to perform at-line measurements spanning from the bioreactor to the downstream processing. QC 20210507

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. QC 20220426

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. QC 20201201

There is a growing recognition of the importance of point-of-care tests (POCTs) for detecting critical neonatal illnesses to reduce the mortality rate in newborns, especially in low-income countries, which account for 98 percent of reported neonatal deaths. Lactate dehydrogenase (LDH) is a marker of cellular damage as a result of hypoxia-ischemia in affected organs. Here, we describe and test a POC LDH test direct from whole blood to provide early indication of serious illness in the neonate. The sample-inresult- out POC platform is specifically designed to meet the needs at resource-limited settings. Plasma is separated from whole blood on filter paper with dried-down reagents for colorimetric reaction, combined with software for analysis using a smartphone. The method was clinically tested in newborns in two different settings. In a clinical cohort of newborns of Stockholm (n = 62) and Hanoi (n = 26), the value of R using Pearson's correlation test was 0.91 (p < 0.01) and the R-2 = 0.83 between the two methods. The mean LDH (+/- SD) for the reference method vs. the POC-LDH was 551 (+/- 280) U/L and 552 (+/- 249) U/L respectively, indicating the clinical value of LDH values measured in minutes with the POC was comparable with standardized laboratory analyses.

Point-of-Care (POC) diagnostics often fail to meet the market requirements of low cost and advanced functionality, and are often limited to lateral flow based serological diagnostics with reduced sensitivity and specificity. We report here on an integrated microfluidic absorbance measurement device fabricated by roll-to-roll (R2R) compatible manufacturing processes, suitable for low cost POC systems. It is a device exclusively made of foils and takes external light from a low cost LED and converts the point light source to a homogeneous light via a foil based optical filter at the bottom of the device. The light is converted to an electrical signal by an amorphous organic semiconductor (OSC) material, integrated with screen-printed carbon finger on top of the device for electrical measurement. As a proof of principle, we demonstrate DNA hybridization assay, where the target DNA is coupled to magnetic beads for absorbance measurement. The device successfully distinguishes between matched and mismatched DNA hybridization and can differentiate between 1 mM, 50 nM and 2.5 nM DNA target concentrations. The inherent characteristics of the substrates and R2R fabrication concept significantly reduce the cost, making it suitable for POC applications at resource-limited settings.

A small subset of HIV-1 infected individuals, the “Elite Controllers” (EC), can control viral replication and restrain progression to immunodeficiency without antiretroviral therapy (ART). In this study, a cross-sectional transcriptomics and targeted proteomics analysis were performed in a well-defined Swedish cohort of untreated EC (n = 19), treatment naïve patients with viremia (VP, n = 32) and HIV-1-negative healthy controls (HC, n = 23). The blood transcriptome identified 151 protein-coding genes that were differentially expressed (DE) in VP compared to EC. Genes like CXCR6 and SIGLEC1 were downregulated in EC compared to VP. A definite distinction in gene expression between males and females among all patient-groups were observed. The gene expression profile between female EC and the healthy females was similar but did differ between male EC and healthy males. At targeted proteomics analysis, 90% (29/32) of VPs clustered together while EC and HC clustered separately from VP. Among the soluble factors, 33 were distinctive to be statistically significant (False discovery rate = 0.02). Cell surface receptor signaling pathway, programmed cell death, response to cytokine and cytokine-mediated signaling seem to synergistically play an essential role in HIV-1 control in EC., Graphical Abstract Image 1, Highlights • The AIDS restriction genes do not have any role in immune control mechanism in EC. • Strong distinction in gene expression between males and females among all patient groups studied. • Multiple pathways play a synergistic role in controlling the viral replication control in EC. • Pathways involving TNFSF/TNFRSF and the immune checkpoint are of importance for immune control in HIV-infected patients. A group of HIV-1 infected individuals termed Elite Controllers (EC) controls viral replication naturally without antiretroviral therapy. In this study, we used an explorative molecular data-first approach using high-throughput transcriptomics and targeted proteomics analysis to understand mechanisms of viral replication control in EC. We observed that EC are more similar to HIV-1 negative healthy controls but a robust gender-specific differentiation was seen in blood transcriptomics profile. Further, combining the gene expression and plasma proteomics profile analysis clearly implicated that several immunological pathways play an important role in HIV-1 control in EC, synergistically through interconnecting molecules.

Flow cytometry is currently the gold standard for analysis of cells in the medical laboratory and biomedical research. Fuelled by the need of point-of-care diagnosis, a significant effort has been made to miniaturize and reduce cost of flow cytometers. However, despite recent advances, current microsystems remain less versatile and much slower than their large-scale counterparts. In this work, an all-silica fibre microflow cytometer is presented that measures fluorescence and scattering from particles and cells. It integrates cell transport in circular capillaries and light delivery by optical fibres. Single-stream cell focusing is performed by Elasto-inertial microfluidics to guarantee accurate and sensitive detection. The capability of this technique is extended to high flow rates (up to 800 µl/min), enabling a throughput of 2500 particles/s. The robust, portable and low-cost system described here could be the basis for a point-of-care flow cytometer with a performance comparable to commercial systems.

A major challenge in evaluating the success of HIV eradication approaches is the need for accurate measurement of persistent HIV during effective antiretroviral therapy (ART). Previous studies have reported that the anti-HIV antibody assay “luciferase immuno-precipitation systems (LIPS)” can distinguish HIV-infected individuals harboring different sizes of the viral reservoirs. We performed antibody profiling of HIV-1 proteomes using LIPS in viremic progressors (n = 38), elite controllers (ECs; n = 19) and patients with fully suppressive long-term antiretroviral therapy (ART) (n = 19) (mean 17 years). IgG was quantified against six HIV-1 fusion proteins: p24, gp41, RT, Tat, integrase and protease. Lower antibody levels to all six-fusion proteins were observed in long-term ART patients compared to viremics (p

[EN] A new advancement in massive DNA-based screening in limited-resource settings is demonstrated through the incorporation of easy-to-fabricate microfluidic chambers on digital versatile discs (DVDs) to perform isothermal recombinase polymerase amplification (RPA) in a microarray format. Standard un-modified DVD discs and commercial drives are used for the low-cost detection method. DNA primers were printed in a microarray format on the polycarbonate surfaces of DVDs with integrated control spots to guarantee the absence of false-negatives and false-positives. The solid-phase amplification assay, including the washing protocols and development reaction, was performed by the dispensation of solutions through the inlet and by controlling the flow-movement by DVD drive centrifugation. The final disc with reaction products was inserted into a DVD player and microarray images were captured and automatically processed. This simple approach was applied for the screening of genetically modified organisms (GMOs) in food samples. The limit of detection was 7 mu g g(-1), which is well below the EU regulation limit for GMOs in food products. Therefore, the only required materials for food safety monitoring were standard store-bought DVDs, plastic chambers, tips, pipettes, an oven, and a standard DVD drive. The proposed strategy allows an integrated microarray system with low manipulation, reduced sample volume, and portability, which are beneficial for low-resource settings., This research has been funded through the projects FP7 Digital sequencing (European Commission), PAID-00-12 (UPV, GVA), FEDER PrometeoII/2014/040 (GVA) and CTQ/2013/45875 R (MINECO). The Spanish Ministry of Education and Science provided S.S.F. with a grant for her PhD studies.

We present a method for PDMS microfabrication of fragile membranes and 3D fluidic networks, using a surface modified water-dissolvable release material, poly(vinyl alcohol), as a tool for handling, transfer and release of fragile polymer microstructures. The method is well suited for the fabrication of complex multilayer microfluidic devices, here shown for a PDMS device with a thin gas permeable membrane and closely spaced holes for vertical interlayer connections fabricated in a single layer. To the authors knowledge, this constitutes the most advanced PDMS fabrication method for the combination of thin, fragile structures and 3D fluidics networks, and hence a considerable step in the direction of making PDMS fabrication of complex microfluidic devices a routine endeavour. QC 20150624

Using the latest innovations in microfabrication technology, 3-dimensional microfluidic cell culture systems have been developed as an attractive alternative to traditional 2-dimensional culturing systems as a model for long-term microscale cell-based research. Most microfluidic systems are based on the embedding of cells in hydrogels. However, physiologically realistic conditions based on hydrogels are difficult to obtain and the systems are often too complicated. We have developed a microfluidic cell culture device that incorporates a biodegradable rigid 3D polymer scaffold using standard soft lithography methods. The device permits repeated high-resolution fluorescent imaging of live cell populations within the matrix over a 4 week period. It was also possible to track cell development at the same spatial location throughout this time. In addition, human primary periodontal ligament cells were induced to produce quantifiable calcium deposits within the system. This simple and versatile device should be readily applicable for cell-based studies that require long-term culture and high-resolution bioimaging. QC 20121029

Acoustophoresis, the ability to acoustically manipulate particles and cells inside a microfluidic channel, is a critical enabling technology for cell-sorting applications. However, one of the major impediments for routine use of acoustophoresis at clinical laboratory has been the reliance on the inherent physical properties of cells for separation. Here, we present a microfluidic-based microBubble-Activated Acoustic Cell Sorting (BAACS) method that rely on the specific binding of target cells to microbubbles conjugated with specific antibodies on their surface for continuous cell separation using ultrasonic standing wave. In acoustophoresis, cells being positive acoustic contrast particles migrate to pressure nodes. On the contrary, air-filled polymer-shelled microbubbles being strong negative acoustic contrast particles migrate to pressure antinodes and can be used to selectively migrate target cells. As a proof of principle, we demonstrate the separation of cancer cell line in a suspension with better than 75% efficiency. Moreover, 100% of the microbubble-cell conjugates migrated to the anti-node. Hence a better upstream affinity-capture has the potential to provide higher sorting efficiency. The BAACS technique expands the acoustic cell manipulation possibilities and offers cell-sorting solutions suited for applications at point of care. Electronic supplementary material The online version of this article (doi:10.1007/s10544-017-0157-4) contains supplementary material, which is available to authorized users.

We study numerically the inertial migration of a single rigid sphere and an oblate spheroid in straight square and rectangular ducts. A highly accurate interface-resolved numerical algorithm is employed to analyse the entire migration dynamics of the oblate particle and compare it with that of the sphere. Similarly to the inertial focusing of spheres, the oblate particle reaches one of the four face-centred equilibrium positions, however they are vertically aligned with the axis of symmetry in the spanwise direction. In addition, the lateral trajectories of spheres and oblates collapse into an equilibrium manifold before ending at the equilibrium positions, with the equilibrium manifold tangential to lines of constant background shear for both sphere and oblate particles. The differences between the migration of the oblate and sphere are also presented, in particular the oblate may focus on the diagonal symmetry line of the duct cross-section, close to one of the corners, if its diameter is larger than a certain threshold. Moreover, we show that the final orientation and rotation of the oblate exhibit a chaotic behaviour for Reynolds numbers beyond a critical value. Finally, we document that the lateral motion of the oblate particle is less uniform than that of the spherical particle due to its evident tumbling motion throughout the migration. In a square duct, the strong tumbling motion of the oblate in the first stage of the migration results in a lower lateral velocity and consequently longer focusing length with respect to that of the spherical particle. The opposite is true in a rectangular duct where the higher lateral velocity of the oblate in the second stage of the migration, with negligible tumbling, gives rise to shorter focusing lengths. These results can help the design of microfluidic systems for bio-applications., Comment: 21 pages, 9 figures