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.

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.

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-based Immunoassays.

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or complicated equipment, making fabrication costly and incompatible with mass production, which is one of the most important preconditions for point-of-care tests (POCT) to be adopted in low-resource settings. This work describes the fabrication process of an acrylic (polymethylmethacrylate, PMMA) device for nanoparticle-conjugated enzymatic immunoassay testing using the computer numerical control (CNC) micromilling technique. The functioning of the microfluidic device is shown by performing an immunoassay to detect a commercial antibody using lysozyme as a model antigen conjugated to 100 nm magnetic nanoparticles. This device integrates a physical staggered restriction of only 5 µm in height, used to capture magnetic microparticles that make up a magnetic trap by placing an external magnet. In this way, the magnetic force on the immunosupport of conjugated nanoparticles is enough to capture them and resist flow drag. This microfluidic device is particularly suitable for low-cost mass production without the loss of precision for immunoassay performance.

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.

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.

Micro-nanoparticles magnetic trap: Toward high sensitivity and rapid microfluidic continuous flow enzyme immunoassay.

In this work, we developed a microfluidic system for immunoassays where we combined the use of magnetic nanoparticles as immunosupport, a microfluidic magnetic trap, and a fluorogenic substrate in continuous flow for detection which, together with the optimization of the functionalization of surfaces to minimize nonspecific interactions, resulted in a detection limit in the order of femtomolar and a total assay time of 40 min for antibiotin antibody detection. A magnetic trap made of carbonyl-iron microparticles packaged inside a 200 µ m square microchannel was used to immobilize and concentrate nanoparticles. We functionalized the surface of the iron microparticles with a silica-polyethylene glycol (PEG) shell to avoid corrosion and unspecific protein binding. A new one-step method was developed to coat acrylic microchannels with an organofunctional silane functionalized with PEG to minimize unspecific binding. A model immunoassay was performed using nanoparticles decorated with biotin to capture antibiotin rabbit Immunoglobulin G (IgG) as target primary antibody. The detection was made using antirabbit IgG labeled with the enzyme alkaline phosphatase as a secondary antibody, and we measured fluorescence with a fluorescence microscope. All steps of the immunoassay were performed inside the chip. A calibration curve was obtained in which a detection limit of 8 pg/ml of antibiotin antibody was quantified. The simplicity of the device and the fact that it is made of acrylic, which is compatible with mass production, make it ideal for Point-Of-Care applications.

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.