Portable platform for leukocyte extraction from blood using sheath-free microfluidic DLD.

Leukocyte count is routinely performed for diagnostic purposes and is rapidly emerging as a significant biomarker for a wide array of diseases. Additionally, leukocytes have demonstrated considerable promise in novel cell-based immunotherapies. However, the direct retrieval of leukocytes from whole blood is a significant challenge due to their low abundance compared to erythrocytes. Here, we introduce a microfluidic-based platform that isolates and recovers leukocytes from diluted whole blood in a single step. Our platform utilizes a novel, sheathless method to initially sediment and focus blood cells into a dense stream while flowing through a tubing before entering the microfluidic device. A hexagonal-shaped structure, patterned at the device's inlet, directs all the blood cells against the channel's outer walls. The focused cells are then separated based on their size using the deterministic lateral displacement (DLD) microfluidic technique. We evaluated various parameters that could influence leukocyte separation, including different focusing structures (assessed both computationally and experimentally), the orientation of the tubing-chip interface, the effects of blood sample hematocrit (dilution), and flow rate. Our device demonstrated the ability to isolate leukocytes from diluted blood with a separation efficiency of 100%, a recovery rate of 76%, and a purity of 80%, while maintaining a cell viability of 98%. The device operates for over 30 min at a flow rate of 2 µL min-1. Furthermore, we developed a handheld pressure controller to drive fluid flow, enhancing the operability of our platform outside of central laboratories and enabling near-patient testing. Our platform can be integrated with downstream cell-based assays and analytical methods that require high leukocyte purity (80%), ranging from cell counting to diagnostics and cell culture applications.

Microfluidic 3D hepatic cultures integrated with a droplet-based bioanalysis unit.

A common challenge in microfluidic cell cultures has to do with analysis of cell function without replacing a significant fraction of the culture volume and disturbing local concentration gradients of signals. To address this challenge, we developed a microfluidic cell culture device with an integrated bioanalysis unit to enable on-chip analysis of picoliter volumes of cell-conditioned media. The culture module consisted of an array of 140 microwells with a diameter of 300 m which were made low-binding to promote organization of cells into 3D spheroids. The bioanalysis module contained a droplet generator unit, 15 micromechanical valves and reservoirs loaded with reagents. Each 0.8 nL droplet contained an aliquot of conditioned media mixed with assay reagents. The use of microvalves allowed us to load enzymatic assay and immunoassay into sequentially generated droplets for detection of glucose and albumin, respectively. As a biological application of the microfluidic device, we evaluated hormonal stimulation and glucose consumption of hepatic spheroids. To mimic physiological processes occurring during feeding and fasting, hepatic spheroids were exposed to pancreatic hormones, insulin or glucagon. The droplet-based bioanalysis module was used to measure uptake or release of glucose upon hormonal stimulation. In the future, we intend to use this microfluidic device to mimic and measure pathophysiological processes associated with hepatic insulin resistance and diabetes in the context of metabolic syndrome.

Millifluidic valves and pumps made of tape and plastic.

There is growing interest in producing micro- and milli-fluidic technologies made of thermoplastic with integrated fluidic control elements that are easy to assemble and suitable for mass production. Here, we developed millifluidic valves and pumps made of acrylic layers bonded with double-sided tape that are simple and fast to assemble. We demonstrate that a layer of pressure-sensitive adhesive (PSA) is flexible enough to be deformed at relatively low pressures. A chemical treatment deposited on specific regions of the PSA prevents it from sticking to the thermoplastic, which enabled us to create three different types of valves in normally open or closed configurations. We characterized different aspects of their performance, their operating pressures, the cut-off pressure values to open or close the valves (for different configurations and sizes), and the flow rate and volume pumped by seven different micropumps. As an application, we implemented a glucose assay with integrated pumps and valves, automatically generating glucose dilutions and reagent mixing. The ability to create polymeric microfluidic control units made with tape paves the way for their mass manufacturing.

Secretion Function Analysis of Ex Vivo Immune Cells in an Integrated Microfluidic Device.

Immune cells play a major role in the development of cancer, from being able to inhibit it by secreting pro-inflammatory mediators, to assist in its development by secreting growth factors, immunosuppressive mediators, and ECM-modifying enzymes. Therefore, the ex vivo analysis of the secretion function of immune cells can be employed as a reliable prognostic biomarker in cancer. However, one limiting factor in current approaches to probe the ex vivo secretion function of cells is their low throughput and the consumption of large quantities of sample. Microfluidics provides a unique advantage, by being able to integrate different components, such as cell culture and biosensors in a monolithic microdevice; it can increase the analytical throughput and leverage it with its intrinsic low sample requirement. Furthermore, the integration of fluid control elements also allows this analysis to be highly automatable, leading to increases in consistency in the results. Here, we describe an approach to analyze the ex vivo secretion function of immune cells using a highly integrated microfluidic device.

On-Chip Analysis of Protein Secretion from Single Cells Using Microbead Biosensors.

The profiling of the effector functions of single immune cells─including cytokine secretion─can lead to a deeper understanding of how the immune system operates and to potential diagnostics and therapeutical applications. Here, we report a microfluidic device that pairs single cells and antibody-functionalized microbeads in hydrodynamic traps to quantitate cytokine secretion. The device contains 1008 microchambers, each with a volume of ∼500 pL, divided into six different sections individually addressed to deliver an equal number of chemical stimuli. Integrating microvalves allowed us to isolate cell/bead pairs, preventing cross-contamination with factors secreted by adjacent cells. We implemented a fluorescence sandwich immunoassay on the biosensing microbeads with a limit of detection of 9 pg/mL and were able to detect interleukin-8 (IL-8) secreted by single blood-derived human monocytes in response to different concentrations of LPS. Finally, our platform allowed us to observe a significant decrease in the number of IL-8-secreting monocytes when paracrine signaling becomes disrupted. Overall, our platform could have a variety of applications for which the analysis of cellular function heterogeneity is necessary, such as cancer research, antibody discovery, or rare cell screening.

Microfluidic systems for the analysis of blood-derived molecular biomarkers.

Biomarkers are relevant indicators of the physiological state of an individual. Although biomarkers can be found in diseased tissue and different biofluids, sampling from blood plasma is relatively easy and less invasive. Among the molecular biomarkers that can be found circulating in plasma are proteins, metabolites, nucleic acids, and exosomes. Some of these plasma-circulating biomarkers are now employed for patient stratification in a broad range of diseases with high sensitivity and specificity and are useful in early diagnosis, initial risk assessment, and therapy selection. However, there is a pressing need to develop novel approaches for biomarker analysis that can be translated into clinical or other settings without complex methodologies or instrumentation. Microfluidics has been touted as a promising technology to carry out this task because it offers high-throughput, automation, multiplexed detection, and portability, possibly overcoming the bottleneck that prevent the translation of novel biomarkers to the point-of-care (POC). Here, we provide a review of the microfluidic systems that have been engineered to detect circulating molecular biomarkers in blood plasma. We also review the different microfluidic approaches for plasma enrichment, which are now being integrated with microfluidic-based biomarker analyzers. Such integration should lead to cost-effective solutions in in vitro diagnostics, with special relevance to POC platforms.

A high-throughput multiplexed microfluidic device for COVID-19 serology assays.

The applications of serology tests to the virus SARS-CoV-2 are diverse, ranging from diagnosing COVID-19, understanding the humoral response to this disease, and estimating its prevalence in a population, to modeling the course of the pandemic. COVID-19 serology assays will significantly benefit from sensitive and reliable technologies that can process dozens of samples in parallel, thus reducing costs and time; however, they will also benefit from biosensors that can assess antibody reactivities to multiple SARS-CoV-2 antigens. Here, we report a high-throughput microfluidic device that can assess antibody reactivities against four SARS-CoV-2 antigens from up to 50 serum samples in parallel. This semi-automatic platform measures IgG and IgM levels against four SARS-CoV-2 proteins: the spike protein (S), the S1 subunit (S1), the receptor-binding domain (RBD), and the nucleocapsid (N). After assay optimization, we evaluated sera from infected individuals with COVID-19 and a cohort of archival samples from 2018. The assay achieved a sensitivity of 95% and a specificity of 91%. Nonetheless, both parameters increased to 100% when evaluating sera from individuals in the third week after symptom onset. To further assess our platform's utility, we monitored the antibody titers from 5 COVID-19 patients over a time course of several weeks. Our platform can aid in global efforts to control and understand COVID-19.

Birefringent optofluidic gratings.

A set of parallel microfluidic channels behaving as a diffraction grating operating in the Raman-Nath regime has been fabricated and studied. The diffraction efficiency of such structure can be tuned by selecting a liquid with a particular refractive index and/or optical anisotropy. Alternatively the optical properties of the liquid can be characterised by measuring the diffraction efficiency and the state of polarization of the diffracted beam. In this work, the microfluidic channels under study have been filled with penicillin molecules dissolved in water. Due to the chirality of the penicillin, the liquid has been found to have circular birefringence of 2.14 × 10-7. The addition of the anisotropic liquid modifies the polarization properties of the microfluidic diffraction grating. The diffraction efficiency of the grating has been characterised for different probe beam wavelengths and states of polarization. Currently the diffraction efficiency of the device is low - 1.7%, but different approaches for its improvement have been discussed.

An affordable 3D-printed positioner fixture improves the resolution of conventional milling for easy prototyping of acrylic microfluidic devices.

We present a simple and low-cost positioner fixture to improve the fabrication resolution of acrylic microchannels using conventional milling machines. The positioner fixture is a mechatronic platform that consists of three piezoelectric actuators assembled in a housing made of 3D printer parts. The upper part of the housing is raised by the simultaneous actuation of the piezoelectric elements and by the deformation of 3D-printed hinge-shaped supports. The vertical positioning (Z-axis) can be controlled with a resolution of 500 nm and an accuracy of ±1.5 µm; in contrast, conventional milling machines can achieve resolutions of 10 to 35 µm. Through simulations, we found that 3D-printed hinges can deform to reach heights up to 27 µm without suffering any mechanical or structural damage. To demonstrate the capabilities of our fixture, we fabricated microfluidic devices with three weir filters that selectively capture microbeads of 3, 6 and 10 µm. We used a similar weir filter design to implement a bead-based immunoassay. Our positioner fixture increases the resolution of conventional milling machines, thus enabling the fast and easy fabrication of thermoplastic fluidic devices that require finer microstructures in their design.

Microfluidic systems for cancer diagnostics.

Although not employed in the clinic as of yet, microfluidic systems are likely to become a key technology for cancer diagnostics and prognosis. Microfluidic devices have been developed for the analysis of various biomarkers including circulating tumor cells, cell-free DNA, exosomes, and proteins, primarily in liquid biopsies such as serum, plasma, and whole blood, avoiding the need for tumor tissue biopsies. Here, we summarize microfluidic technological advances that are used in cancer diagnosis, prognosis, and to monitor its progression and recurrence, that will likely lead to personalized therapies. In some cases, integrated microfluidic technologies, coupled with biosensors, are proving to be more sensitive and precise in the detection of cancer biomarkers than conventional assays. Based on the current state-of-the-art and the rapid progress over the past decade, we also briefly discuss the next evolutionary steps that these technologies are likely to take.

A versatile microfluidic device for multiple ex vivo/in vitro tissue assays unrestrained from tissue topography.

Precision-cut tissue slices are an important in vitro system to study organ function because they preserve most of the native cellular microenvironments of organs, including complex intercellular connections. However, during sample manipulation or slicing, some of the natural surface topology and structure of these tissues is lost or damaged. Here, we introduce a microfluidic platform to perform multiple assays on the surface of a tissue section, unhindered by surface topography. The device consists of a valve on one side and eight open microchannels located on the opposite side, with the tissue section sandwiched between these two structures. When the valve is actuated, eight independent microfluidic channels are formed over a tissue section. This strategy prevents cross-contamination when performing assays and enables parallelization. Using irregular tissues such as an aorta, we conducted multiple in vitro and ex vivo assays on tissue sections, including short-term culturing, a drug toxicity assay, a fluorescence immunohistochemistry staining assay, and an immune cell assay, in which we observed the interaction of neutrophils with lipopolysaccharide (LPS)-stimulated endothelium. Our microfluidic platform can be employed in other disciplines, such as tissue physiology and pathophysiology, morphogenesis, drug toxicity and efficiency, metabolism studies, and diagnostics, enabling the conduction of several assays with a single biopsy sample.

Liquid refractive index measured through a refractometer based on diffraction gratings.

We developed two versions of refractometers to measure the refractive index of liquids. One refractometer comprises a glass cell with a surface relief grating on the inner face of one of its walls, while the other one is a microfluidic channel in the form of serpentine that behaves as a grating. Measurements of the liquid refractive index were performed by sensing the first order intensity. Several liquids have been used including an organic one. Calibration plots are shown.

Facile assembly of an affordable miniature multicolor fluorescence microscope made of 3D-printed parts enables detection of single cells.

Fluorescence microscopy is one of the workhorses of biomedical research and laboratory diagnosis; however, their cost, size, maintenance, and fragility has prevented their adoption in developing countries or low-resource settings. Although significant advances have decreased their size, cost and accessibility, their designs and assembly remain rather complex. Here, inspired on the simple mechanism from a nut and a bolt, we report the construction of a portable fluorescence microscope that operates in bright-field mode and in three fluorescence channels: UV, green, and red. It is assembled in under 10 min from only six 3D printed parts, basic electronic components, a microcomputer (Raspberry Pi) and a camera, all of which can be readily purchased in most locations or online for US $122. The microcomputer was programmed in Python language to capture time-lapse images and videos. Resolution and illumination conditions of the microscope were characterized, and its performance was compared with a high-end fluorescence microscope in bright-field and fluorescence mode. We demonstrate that our miniature microscope can resolve and track single cells in both modes. The instructions on how to assemble the microscope are shown in a video, and the software to control it and the design files of the 3D-printed parts are freely available online. Our portable microscope is ideal in applications where space is at a premium, such as lab-on-a-chips or space missions, and can find applications in basic and clinical research, diagnostics, telemedicine and in educational settings.

Automated Droplet-Based Microfluidic Platform for Multiplexed Analysis of Biochemical Markers in Small Volumes.

The ability to detect multiple analytes in a small sample volume has significance for numerous areas of research, including organs-on-chip, small animal experiments, and neonatology. The objective of this study was to develop an automated microfluidics platform for multiplexed detection of analytes in microliter sample volumes. This platform employed computer-controlled microvalves to create laminar co-flows of sample and assay reagent solutions. It also contained valve-regulated cross-junction for discretizing sample/reagent mixtures into water-in-oil droplets. Microfluidic automation allowed us to control parameters related to frequency of droplet generation and the number of droplets of the same composition, as well as the size of droplets. Each droplet represented an individual enzymatic assay carried out in a sub-nanoliter (0.8 nL) volume reactor. An enzymatic reaction involving target analyte and assay reagents produced colorimetric or fluorescent signals in droplets. Importantly, intensity of optical signal was proportional to the concentration of analyte in question. This microfluidic bioanalysis platform was used in conjunction with commercial "mix-detect" assays for glucose, total bile acids, and lactate dehydrogenase (LDH). After characterizing these assays individually, we demonstrated sensitive multiplexed detection of three analytes from as little as 3 µL. In fact, this volume was sufficient to generate multiple repeat droplets for each of the three biochemical assays as well as positive control droplets, confirming the quality of assay reagents and negative control droplets to help with background subtraction. One potential application for this microfluidic bioanalysis platform involves sampling cell-conditioned media in organ-on-chip devices. To highlight this application, hepatocyte spheroids were established in microfluidic devices, injured on-chip by exposure to lipotoxic agent (palmitate), and then connected to the bioanalysis module for daily monitoring of changes in cytotoxicity (LDH), energy metabolism (glucose), and liver function (total bile acids). Microfluidic in-droplet assays revealed increased levels of LDH as well as reduction in bile acid synthesis-results that were consistent with hepatic injury. Importantly, these experiments highlighted the fact that in-droplet assays were sufficiently sensitive to detect changes in functional output of a relatively small (∼100) number of hepatocyte spheroids cultured in a microfluidic device. Moving forward, we foresee increasing the multiplexing capability of this technology and applying this platform to other biological/medical scenarios where detection of multiple analytes from a small sample volume is desired.

Dynamic Generation of Concentration- and Temporal-Dependent Chemical Signals in an Integrated Microfluidic Device for Single-Cell Analysis.

Intracellular signaling pathways are affected by the temporal nature of external chemical signaling molecules such as neurotransmitters or hormones. Developing high-throughput technologies to mimic these time-varying chemical signals and to analyze the response of single cells would deepen our understanding of signaling networks. In this work, we introduce a microfluidic platform to stimulate hundreds of single cells with chemical waveforms of tunable frequency and amplitude. Our device produces a linear gradient of 9 concentrations that are delivered to an equal number of chambers, each containing 492 microwells, where individual cells are captured. The device can alternate between the different stimuli concentrations and a control buffer, with a maximum operating frequency of 33 mHz that can be adjusted from a computer. Fluorescent time-lapse microscopy enables to obtain hundreds of thousands of data points from one experiment. We characterized the gradient performance and stability by staining hundreds of cells with calcein AM. We also assessed the capacity of our device to introduce periodic chemical stimuli of different amplitudes and frequencies. To demonstrate our device performance, we studied the dynamics of intracellular Ca2+ release from intracellular stores of HEK cells when stimulated with carbachol at 4.5 and 20 mHz. Our work opens the possibility of characterizing the dynamic responses in real time of signaling molecules to time-varying chemical stimuli with single cell resolution.

An Affordable and Portable Thermocycler for Real-Time PCR Made of 3D-Printed Parts and Off-the-Shelf Electronics.

The polymerase chain reaction (PCR) is a sought-after nucleic acid amplification technique used in the detection of several diseases. However, one of the main limitations of this and other nucleic acid amplification assays is the complexity, size, maintenance, and cost of their operational instrumentation. This limits the use of PCR applications in settings that cannot afford the instruments but that may have access to basic electrical, electronic, and optical components and the expertise to build them. To provide a more accessible platform, we developed a low-cost, palm-size, and portable instrument to perform real-time PCR (qPCR). The thermocycler leverages a copper-sheathed power resistor and a computer fan, in tandem with basic electronic components controlled from a single-board computer. The instrument incorporates a 3D-printed chassis and a custom-made fluorescence optical setup based on a CMOS camera and a blue LED. Results are displayed in real-time on a tablet. We also fabricated simple acrylic microdevices consisting of four wells (2 µL in volume each) where PCR reactions take place. To test our instrument, we performed qPCR on a series of cDNA dilutions spanning 4 orders of magnitude, achieving similar limits of detection as those achieved by a benchtop thermocycler. We envision our instrument being utilized to enable routine monitoring and diagnosis of certain diseases in low-resource areas.

Pressure-actuated monolithic acrylic microfluidic valves and pumps.

In this article, we describe a microfluidic device with embedded valves and pumps made exclusively of layers of acrylic glass. Flat acrylic sheets are carved out with a micromilling machine and bonded together by solvent bonding. The working principle of the valves is based on a thin flexible membrane (≈100 µm) machined on one acrylic sheet and actuated with pneumatic pressure. A completely closed valve resists a pressure difference of ≈17 kPa (≈2.5 psi), and when open, it can sustain flow rates of up to 100 µL s-1. Pumping is achieved by combining two valves and a pumping chamber in series, which is also based on the bending of a thin acrylic membrane. The maximum flow rate obtained with this pumping mechanism is 20 µL min-1. Acrylic is a popular rigid thermoplastic because it is inexpensive, making it ideal for mass production of disposable devices, and also because it has demonstrated compatibility with different biochemical assays. The physical and optical properties it shares with other thermoplastics could lead to this material being implemented for similar valves and pumps. As a proof-of-concept of our technology, we implemented a controlled cell-staining assay in two parallel incubation chambers integrating four valves and one pump into one device. Our monolithic acrylic valves can enable the mass production of disposable microfluidic devices that require fluid control with pressure-actuated valves and aid in the automation of biochemical assays.

Sessile droplets for chemical and biological assays.

Sessile droplets are non-movable droplets spanning volumes in the nL-to-µL range. The sessile-droplet-based platform provides a paradigm shift from the conventional, flow-based lab-on-a-chip philosophy, yet offering similar benefits: low reagent/sample consumption, high throughput, automation, and most importantly flexibility and versatility. Moreover, the platform relies less heavily on sophisticated fabrication techniques, often sufficient with a hydrophobic substrate, and no pump is required for operation. In addition, exploiting the physical phenomena that naturally arise when a droplet evaporates, such as the coffee-ring effect or Marangoni flow, can lead to fascinating applications. In this review, we introduce the physics of droplets, and then focus on the different types of chemical and biological assays that have been implemented in sessile droplets, including analyte concentration, particle separation and sorting, cell-based assays, and nucleic acid amplification. Finally, we provide our perspectives on this unique micro-scale platform.

Massive Parallel Analysis of Single Cells in an Integrated Microfluidic Platform.

New tools that facilitate the study of cell-to-cell variability could help uncover novel cellular regulation mechanisms. We present an integrated microfluidic platform to analyze a large number of single cells in parallel. To isolate and analyze thousands of individual cells in multiplexed conditions, our platform incorporates arrays of microwells (7 pL each) in a multilayered microfluidic device. The device allows the simultaneous loading of cells into 16 separate chambers, each containing 4640 microwells, for a total of 74 240 wells per device. We characterized different parameters important for the operation of the microfluidic device including flow rate, solution exchange rate in a microchamber, shear stress, and time to fill up a single microwell with molecules of different molecular weight. In general, after ∼7.5 min of cell loading our device has an 80% microwell occupancy with 1-4 cells, of which 36% of wells contained a single cell. To test the functionality of our device, we carried out a cell viability assay with adherent and nonadherent cells. We also studied the production of neutrophil extracellular traps (NETs) from single neutrophils isolated from peripheral blood, observing the existence of temporal heterogeneity in NETs production, perhaps having implications in the type of the neutrophil response to an infection or inflammation. We foresee our platform will have a variety of applications in drug discovery and cellular biology by facilitating the characterization of phenotypic differences in a monoclonal cell population.

Simple scaling laws for the evaporation of droplets pinned on pillars: Transfer-rate- and diffusion-limited regimes.

The evaporation of droplets can give rise to a wide range of interesting phenomena in which the dynamics of the evaporation are crucial. In this work, we find simple scaling laws for the evaporation dynamics of axisymmetric droplets pinned on millimeter-sized pillars. Different laws are found depending on whether evaporation is limited by the diffusion of vapor molecules or by the transfer rate across the liquid-vapor interface. For the diffusion-limited regime, we find that a mass-loss rate equal to 3/7 of that of a free-standing evaporating droplet brings a good balance between simplicity and physical correctness. We also find a scaling law for the evaporation of multicomponent solutions. The scaling laws found are validated against experiments of the evaporation of droplets of (1) water, (2) blood plasma, and (3) a mixture of water and polyethylene glycol, pinned on acrylic pillars of different diameters. These results shed light on the macroscopic dynamics of evaporation on pillars as a first step towards the understanding of other complex phenomena that may be taking place during the evaporation process, such as particle transport and chemical reactions.