Extracellular vesicles (EVs), especially the exosome-sized subset are increasingly exploited as minimally invasive cancer biomarkers. These small vesicles are abundant in biofluids and play crucial roles in intercellular communication and disease progression by transporting bioactive molecules. Exosomes offer distinct diagnostic and prognostic advantages over traditional cancer biomarkers, but purifying and extracting exosomes from blood remains challenging. There is a need to simply and cost-effectively isolate exosomes from milliliter quantities of whole blood for transcriptional and other omics-based research. Addressing this gap, we propose a microfluidic cartridge, the EV-Blade, for size and affinity-based purification of exosomes on a centrifugal microfluidic platform. We demonstrate a method to automate exosome purification from whole blood samples on a single microfluidic cartridge. The EV-Blade system combines blood centrifugation, plasma filtration for EV size selection and immunomagnetic capture using functionalized superparamagnetic nanoparticles targeting CD9, CD63, and CD81 exosomal surface proteins. We report on the device performance, purity of exosome recovery and the quality of RNA collected following on-chip EV lysis. We use this automated method to detect relevant long coding and non-coding RNA transcripts in circulating blood exosomes, showcasing the EV-Blade for use in cancer patient risk stratification. The system presented herein represents a significant advancement in exosome purification, offering a robust and automated platform for liquid biopsy-based cancer research and clinical applications. This innovation holds promise for cancer diagnosis, prognosis, and monitoring through non-invasive biomarkers.

This paper describes the development of an integrated assay for the purification of adeno-associated virus (AAV) particles from crude lysate using a microfluidic cartridge and a centrifugal platform that enables pneumatic actuation of liquids during rotation. The cartridge features POROS™ CaptureSelect™ AAVX Affinity Resin as a solid-phase extraction (SPE) matrix and a layout that is compatible with workflow automation and low-volume sample processing. The integrated process takes ∼30 to 60 min depending on the input volume and comprises 22 consecutive steps, starting with crude lysate and ending with an AAV sample conditioned for downstream analysis or long-term storage. Numerical simulations, conducted as part of the design validation, reveal uniform flow across the SPE matrix for both in-plane and out-of-plane orientations. The performance of the system is demonstrated using AAV8 with volumes ranging from 50 to 500 μL. The on-chip purification process generally shows higher extraction efficiency than a benchmark procedure performed manually using spin columns. The capacity to remove plasmid DNA is validated using samples with calibrated spike-in concentration that were analyzed by qPCR, suggesting depletion of >99.99%. Biological activity of purified AAV particles was assessed in a transduction assay involving human embryonic kidney cells and recombinant AAV2 expressing green fluorescent protein. The platform's compatibility with low-volume production addresses gaps in current gene therapy manufacturing pipelines, underscoring its potential to streamline quality assurance protocols, reduce manual intervention, and accelerate scalable AAV production.

Microfluidic techniques for high-throughput encapsulation are powerful tools in single-cell analytics and cytokine profiling. Inertial focusing microfluidics is widely used to align particles in uniform sequences, enhancing encapsulation efficiency. However, on-chip sample dilution strategies to further optimize efficiency remain largely unexplored in deterministic encapsulation approaches, both experimentally and through theoretical modeling. Here, we present a high-yield microparticle encapsulation method that combines inertial and hydrodynamic focusing to enable precise tuning of microparticle spacing and modulation of capture efficiency, thereby offering enhanced operational flexibility for controlled particle encapsulation. We first investigate the microparticle self-ordering behavior within the spiral loop and characterize both flow dynamics and droplet formation regimes. By varying the sheath-to-sample flow rate ratio from 0 to 2, we observe that higher ratios increase the interparticle spacing and shift particles closer to the channel wall. These trends align with both analytical modeling and 3D numerical simulations. Notably, at higher sheath flow ratios (e.g., 1 and 2), single-particle encapsulation exceeds 76%, significantly surpassing Poisson distribution predictions. Moreover, single-cell capture efficiency exceeds 60% under these conditions. In co-encapsulation experiments, we achieved a one-cell-multiple-beads co-encapsulation efficiency near 40%, marking a significant improvement over the Poisson limit. For single-cell applications, we performed co-encapsulation of THP-1 monocytes and streptavidin-coated magnetic beads for TNF-α cytokine detection following lipopolysaccharide stimulation. Cytokine secretion was successfully detected at the single-cell level in both aqueous droplets and alginate hydrogels. We anticipate that this method will offer a promising platform for probing cell-cell interactions and immune responses at single-cell resolution.

Organs-on-chips (OoCs) are miniaturized devices for culturing and stimulating living cells within microscopic fluidic environments to create minimal organ surrogates. For the last decade, a diverse range of organ models were reconstructed at the micron scale to support the investigation of fundamental biological processes. By more accurately replicating the pathophysiology of human health, OoCs can serve as alternatives to conventional 2D cell culture and animal models for drug testing. However, moving from a research prototype to a commercially viable model system requires consideration of materials and manufacturing methods to simultaneously sustain high biological fidelity of organ mimicries, while enabling fabrication at scale. This review synthesizes recent advancements in that regard: We provide an overview of materials exhibiting relevant functional properties, their associated manufacturing methods, considerations of metrology and biocompatibility, and showcase representative examples of disruptive OoCs technologies currently integrated in the drug development pipeline to accelerate the discovery of safer and more effective therapeutics.

Microfluidic systems are increasingly utilized in preclinical research, however, developing platforms that are compatible with large-scale manufacturing remains a critical challenge. In this regard, we show that thermoplastic elastomer (TPE)-based microfluidic chips could be a viable alternative to traditional poly(dimethylsiloxane) (PDMS) devices for drug testing applications. Microfluidic concentration gradient generators are essential components in these systems, enabling local spatial and temporal control of biochemical stimuli for applications such as cellular response studies and drug screening. TPEs offer key advantages, especially, optical transparency, flexibility, and compatibility with high-throughput fabrication processes compared to PDMS, thereby enhancing commercialization potential. This study demonstrates the design and fabrication of TPE-based microfluidic devices incorporating concentration gradient generators that allow precise control over solute distributions. Gradient formation was validated through numerical modeling and co-flow experiments using fluorescent dyes. The platform supported the co-culture of human lung fibroblast cells and human colon adenocarcinoma cells (HT-29), which exhibited coordinated responses to gradients of fetal bovine serum (FBS) and sepantronimum bromide (YM-155). Performance benchmarks against PDMS chips demonstrated comparable functionality, underscoring the potential of TPE-based devices for future scalable and cost-effective drug testing and cellular assays.

Sepsis is a life-threatening organ dysfunction due to a dysfunctional response to infection. Delays in diagnosis have substantial impact on survival. Herein, blood samples from 586 in-house patients with suspected sepsis are used in conjunction with machine learning and cross-validation to define a six-gene expression signature of immune cell reprogramming, termed Sepset, to predict clinical deterioration within the first 24 h (h) of clinical presentation. Prediction accuracy (~90% in early intensive care unit (ICU) and 70% in emergency room patients) is validated in 3178 patients from existing independent cohorts. A RT-PCR-based Sepset detection test shows a 94% sensitivity in 248 patients to predict worsening of the sequential organ failure assessment scores within the first 24 h. A stand-alone centrifugal microfluidic instrument that automates whole-blood Sepset classifier detection is tested, showing a sensitivity of 92%, and specificity of 89% in identifying the risk of clinical deterioration in patients with suspected sepsis.

A novel approach to protein quantification utilizing a microfluidic platform activated by a magnetic assembly of functionalized magnetic beads around soft magnetic capture centers is presented. Functionalized magnetic beads, known for their high surface area and facile manipulation under external magnetic fields, are injected inside microfluidic channels and immobilized magnetically on the surface of glass-coated soft magnetic microwires placed along the symmetry axis of these channels. A fluorescent (Cy5) immunomagnetic sandwich ELISA is then performed by sequentially flowing the sample and all necessary reagents in the microfluidic channels. Direct protein quantification is performed by magnetically releasing the beads from the microwire and evaluating their fluorescence intensity with the help of a miniature (microfluidic-based) flow cytometer. Measurements of ICAM-1 protein concentration in human blood plasma samples confirm the feasibility of the approach through extensive performance benchmarking. The automation and multiplexing capabilities of the proposed platform further demonstrate its potential for protein quantification in point-of-care settings using microfluidics and miniature flow cytometry instruments.

Acute respiratory tract infections (ARTIs) are caused by sporadic or pandemic outbreaks of viral or bacterial pathogens, and continue to be a considerable socioeconomic burden for both developing and industrialized countries alike. Diagnostic methods and technologies serving as the cornerstone for disease management, epidemiological tracking, and public health interventions are evolving continuously to keep up with the demand for higher sensitivity, specificity and analytical throughput. Microfluidics is becoming a key technology in these developments as it allows for integrating, miniaturizing and automating bioanalytical assays at an unprecedented scale, reducing sample and reagent consumption and improving diagnostic performance in terms of sensitivity, throughput and response time. In this article, we describe relevant ARTIs-pneumonia, influenza, severe acute respiratory syndrome, and coronavirus disease 2019-along with their pathogenesis. We provide a summary of established methods for disease diagnosis, involving nucleic acid amplification techniques, antigen detection, serological testing as well as microbial culture. This is followed by a short introduction to microfluidics and how flow is governed at low volume and reduced scale using centrifugation, pneumatic pumping, electrowetting, capillary action, and propagation in porous media through wicking, for each of these principles impacts the design, functioning and performance of diagnostic tools in a particular way. We briefly cover commercial instruments that employ microfluidics for use in both laboratory and point-of-care settings. The main part of the article is dedicated to emerging methods deriving from the use of miniaturized, microfluidic systems for ARTI diagnosis. Finally, we share our thoughts on future perspectives and the challenges associated with validation, approval, and adaptation of microfluidic-based systems.

Droplet digital polymerase chain reaction (ddPCR) stands out as a highly sensitive diagnostic technique that is gaining traction in infectious disease diagnostics due to its ability to quantitate very low numbers of viral gene copies. By partitioning the sample into thousands of droplets, ddPCR enables precise and absolute quantification without relying on a standard curve. However, current ddPCR systems often exhibit relatively low levels of integration, and the analytical process remains dependent on elaborate workflows for up-front sample preparation. Here, we introduce a fully-integrated system seamlessly combining viral lysis, RNA extraction, emulsification, reverse transcription (RT) ddPCR, and fluorescence readout in a sample-to-answer format. The system comprises a disposable microfluidic cartridge housing buffers and reagents required for the assay, and a centrifugal platform that allows for pneumatic actuation of liquids during rotation, enabling automation of the workflow. Highly monodisperse droplets (∼50 μm in diameter) are produced using centrifugal step emulsification and automatically transferred to an integrated heating module for target amplification. The platform is equipped with a miniature fluorescence imaging system enabling on-chip read-out of droplets after RT-ddPCR. We demonstrate sample-to-answer detection of SARS-CoV-2 N and E genes, along with RNase P endogenous reference, using hydrolysis probes and multiplexed amplification within single droplets for concentrations as low as 0.1 copy per μL. We also tested 14 nasopharyngeal swab specimens from patients and were able to distinguish positive and negative SARS-CoV-2 samples with 100% accuracy, surpassing results obtained by conventional real-time amplification.

Sepsis is the bodys dysfunctional response to infection associated with organ failure. Delays in diagnosis have a substantial impact on survival. Herein, samples from 586 in-house patients were used in conjunction with machine learning and cross-validation to narrow a gene expression signature of immune cell reprogramming to predict clinical deterioration in patients with suspected sepsis within the first 24 hours (h) of clinical presentation using just six genes (Sepset). The accuracy of the test (~90% in early intensive care unit (ICU) and 70% in emergency room patients) was validated in 3,178 patients from existing independent cohorts. A real-time reverse transcriptase polymerase chain reaction (RT-PCR)-based test was shown to have a 98% sensitivity in >230 patients to predict worsening of the sequential organ failure scores or admission to the ICU within the first 24 h following Sepset detection. A stand-alone centrifugal microfluidic instrument that integrates the entire automated workflow for detection of the Sepset classifier in whole blood using digital droplet PCR was developed and tested. This PREcision meDIcine for CriTical care (PREDICT) system had a high sensitivity of 92%, specificity of 89%, and an overall accuracy of 88% in identifying the risk of imminent clinical deterioration in patients with suspected sepsis. ### Competing Interest Statement Conflicts of Interest REWH is an inventor of the Sepset signature that has been patented in 17 countries (e.g. US patent 11,851,717 issued Dec 26, 2023) and is CEO and a shareholder of Asep Medical and its subsidiary Sepset BioSciences Inc. that have licensed in these patents and are actively commercially developing sepsis diagnostics. REWH also has a contract from Sepset Biosciences for development of diagnostic assays for adult sepsis. PGYZ and EFH are employees of Sepset Biosciences Inc. and/or Asep Medical. ### Clinical Protocols ### Funding Statement Funding statement included in manuscript ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Unity Health Toronto REB approval I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Previously published data can be obtained from GEO. Data from internal validation cohort can be obtained upon request

Automated on-chip sample-to-answer workflow for multiplexed colorimetric detection of foodborne viruses using an embedded polymer micropillar array for read-out.

This paper describes on-the-fly physical property changes of aqueous two-phase systems (ATPS) in microfluidic devices. The properties and phases of the ATPS are modulated on-demand by using a centrifugal microfluidic device filled with poly(ethylene glycol) (PEG) and dextran (DEX) solutions. By use of the centrifugal force and active pneumatic controls provided by a centrifugal microfluidic platform (CMP), PEG-DEX mixtures are manipulated and processed inside simple thermoplastic microfluidic devices. First, we experimentally demonstrate an on-chip ATPS transition from two phases to a single phase and vice versa by dynamically changing the concentration of the solution to bring ATPS across the binodal curve. We also demonstrate a density modulation scheme by introducing an ATPS solution mixed with sodium diatrizoate hydrate, which allows to increase the liquid density. By adding precisely metered volumes of water, we spontaneously change the density of the solution on the CMP and show that density marker microbeads fall into the solution according to their corresponding densities. The measured densities of ATPS show a good agreement with densities of microbeads and analytical plots. The results presented in this paper highlight the tremendous potential of CMPs for performing complex on-chip processing of ATPS. We anticipate that this method will be useful in applications such as microparticle-based plasma protein analysis and blood cell fractionation.

We investigate the formation of suspended magnetic nanoparticle (MNP) assemblies (M-clouds) and their use for in situ bacterial capture and DNA extraction. M-clouds are obtained as a result of magnetic field density variations when magnetizing an array of micropillars coated with a soft ferromagnetic NiP layer. Numerical simulations suggest that the gradient in the magnetic field created by the pillars is four orders of magnitude higher than the gradient generated by the external magnets. The pillars therefore serve as the sole magnetic capture sites for MNPs which accumulate on opposite sides of each pillar facing the magnets. Composed of loosely aggregated MNPs, the M-cloud can serve as a porous capture matrix for target analyte flowing through the array. The concept is demonstrated by using a multifunctional M-cloud comprising immunomagnetic NPs (iMNPs) for capture of Escherichia coli O157:H7 from river water along with silica-coated NPs for subsequent isolation and purification of microbial DNA released upon bacterial lysis. Confocal microscopy imaging of fluorescently labeled iMNPs and E. coli O157:H7 reveals that bacteria are trapped in the M-cloud region between micropillars. Quantitative assessment of in situ bacterial capture, lysis and DNA isolation using real-time polymerase chain reaction shows linear correlation between DNA output and input bacteria concentration, making it possible to confirm E. coli 0157:H7 at 10(3) cells per mL. The M-cloud method further provides one order of magnitude higher DNA output concentrations than incubation of the sample with iMNPs in a tube for an equivalent period of time (e.g., 10 min). Results from assays performed in the presence of Listeria monocytogenes (at 10(6) cells per mL each) suggest that non-target organisms do not affect on-chip E. coli capture, DNA extraction efficiency and quality of the eluted sample.

Fractionating whole blood and separating its constituent components one from another is an essential step in many clinical applications. Currently blood sample handling and fractionation processes remain a predominantly manual task that require well-trained operators to produce reliable and reproducible results. Herein, we demonstrate an advanced on-chip whole human blood fractionation and cell isolation process combining (i) an aqueous two-phase system (ATPS) to create complex separation layers with (ii) a centrifugal microfluidic platform (PowerBlade) with active pneumatic pumping to control and automate the assay. We use a polyethylene glycol (PEG) and dextran (DEX) mixture as the two-phase density gradient media and our automated centrifugal microfluidic platform to fractionate blood samples. Different densities of precisely tuned PEG-DEX solutions were tested to match each of the cell types typically targeted during blood fractionation applications. By employing specially designed microfluidic devices, we demonstrate the automation of the following steps: loading of a whole blood sample on-chip, layering of the blood on the ATPS solution, blood fractionation, precise radial repositioning of the fractionated layers, and finally extraction of multiple, selected fractionated components. Fractionation of up to six distinct layers is shown: platelet-rich plasma, buffy coat, PEG, DEX with neutrophils, red blood cells (RBCs) and high density gradient media (HDGM). Furthermore, through controlled dispensing of HDGM to the fractionation chamber, we show that each of the fractionated layers can be repositioned radially, on-the-fly, without disturbing the interfaces, allowing precise transfer of target fractions and cell types into external vials via a chip-to-world interface. Cell counting analysis and cell viability studies showed equivalence to traditional, manual methods. An overall cell viability greater than 90% of extracted cells demonstrates that the proposed approach is suitable for cell isolation applications. This proof-of-principle demonstration highlights the utility of the proposed system for automated whole blood fractionation and isolation for blood cell applications. We anticipate that the proposed approach will be a useful tool for many clinical applications such as standard cell isolation procedures and other bioanalytical assays (e.g., circulating tumor cells, and cell and gene therapy).

We present here a new method for controlling the droplet size in step emulsification processes on a centrifugal microfluidic platform, which, in addition to the centrifugal force, uses pneumatic actuation for fluid displacement. We highlight the importance of the interplay between buoyancy effects and the flow rate at the step junction, and provide a simple analytical model relating these two quantities to the size of the droplets. Numerical models as well as experiments with water-in-oil emulsions are performed in support of the proposed model.

The development of technology for the rapid, automated identification of bacterial culture isolates can help regulatory agencies to shorten response times in food safety surveillance, compliance, and enforcement as well as outbreak investigations. While molecular methods such as polymerase chain reaction (PCR) enable the identification of microbial organisms with high sensitivity and specificity, they generally rely on sophisticated instrumentation and elaborate workflows for sample preparation with an undesirably high level of hands-on engagement. Herein, we describe the design, operation and performance of a lab-on-a-chip system integrating thermal lysis, PCR amplification and microarray hybridization on the same cartridge. The assay is performed on a centrifugal microfluidic platform that allows for pneumatic actuation of liquids during rotation, making it possible to perform all fluidic operations in a fully-automated fashion without the need for integrating active control elements on the microfluidic cartridge. The cartridge, which is fabricated from hard and soft thermoplastic polymers, is compatible with high-volume manufacturing (e.g., injection molding). Chip design and thermal interface were both optimized to ensure efficient heat transfer and allow for fast thermal cycling during the PCR process. The integrated workflow comprises 14 steps and takes less than 2 h to complete. The only manual steps are related to loading of the sample and reagents on the cartridge as well as fluorescence imaging of the microarray. On-chip lysis and PCR amplification both provided results comparable to those obtained by bench-top instrumentation. The microarray, incorporating a panel of oligonucleotide probes for multiplexed detection of seven enterohemorrhagic E. coli priority serotypes, was implemented on a cyclic olefin copolymer substrate using a novel activation scheme that involves the conversion of hydroxyl groups (derived from oxygen plasma treatment) into reactive cyanate ester using cyanogen bromide. On-chip hybridization was demonstrated in a non-quantitative fashion using fluorescently-labelled gene markers for E. coli O157:H7 (rfbO157, eae, vt1, and vt2) obtained through a multiplexed PCR amplification step.

We describe the use of periodic micropillar arrays, produced from cyclic olefin copolymer using high-fidelity microfabrication, as templates for colorimetric DNA detection. The assay involves PCR-amplified gene markers for E. coli O157:H7 (rfbO157, eae, vt1, and vt2) incorporating a detectable digoxigenin label, which is revealed through an immunoenzymatic process following hybridization with target-specific oligonucleotide capture probes. The capacity of micropillar arrays to induce wicking is used to distribute and confine capture probes with spatial control, making it possible to achieve a uniform signal while allowing multiple, independent probes to be arranged in close proximity on the same substrate. The kinetic profile of color pigment formation on the surface was followed using absorbance measurements, showing maximum signal increase between 20 and 60 min of reaction time. The relationship between microstructure and colorimetric signal was investigated through variation of geometric parameters, such as pitch (10-50 mu m), pillar diameter (5-40 mu m), and height (16-48 mu m). Our findings suggest that signal intensity is largely influenced by the edges of the pillars and less by their height such that it deviates from a linear relationship when both aspect ratio and pillar density become very high. A theoretical model used to simulate the changes in surface composition at the molecular level suggests that differences in the temporal and spatial accumulation of assay components account for this observation.

This paper describes the development of an on-chip nucleic acid (NA) extraction assay from whole blood using a centrifugal microfluidic platform that allows for pneumatic actuation of liquids during rotation. The combination of pneumatic and centrifugal forces makes it possible to perform fluidic operations without the need for integrating active control elements on the microfluidic cartridge. The cartridge is fabricated from thermoplastic polymers (e.g., Zeonor 1060R) and features a simple design that is compatible with injection molding. In addition, the cartridge is interfaced with two external vials for off-chip storage of the blood sample and retrieval of the eluted NA solution, respectively. On-chip capture of NAs is performed using an embedded solid-phase extraction matrix composed of commercial glass microfiber filters (Whatman GF/D and GF/F). The yield of the automated, on-chip extraction protocol, determined by measuring absorbance at 260 nm, is comparable to some of the best manually operated kits (e.g., Qiagen QIAamp DNA Mini Kit) while providing low assay-to-assay variability due to the high level of control provided by the platform for each processing step. The A260/A280 and A260/A230 ratios of the absorbance spectra also reveal that protein contamination of the sample is negligible. The capability of the pneumatic platform to circulate air flux through the microfluidic conduit was used to dry leftover ethanol residues retained in the capture matrix during washing. This method, applied in combination with localized heating, proved effective for reducing ethanol contamination in eluted samples from ∼12% to 1% (v/v).

This review is an account of centrifugal microfluidic systems that use various actuation strategies in addition to intrinsic centrifugal forces to accurately regulate the motion of fluids during rotation. Platforms that integrate active methods of pumping and flow control render centrifugal microfluidics more versatile as they facilitate integration and process automation by enabling (or improving the reliability of) important fluidic functions, such as metering, aliquoting, valving, flow switching, mixing, and inward pumping. Principles and working mechanisms underlying these strategies are described in the context of recent trends in instrument design and development where centrifugal platforms have been equipped with pneumatic, magnetic or electromechanical actuators serving as pumping and valving elements. The potential of these platforms to perform complex bioanalytical assays in an automated fashion is illustrated by several examples, which include on-chip preparation of aliquot libraries, nucleic acid purification, amplification and analysis as well as blood separation.