Label-free separation of peripheral blood mononuclear cells from whole blood by gradient acoustic focusing.

Efficient techniques for separating target cells from undiluted blood are necessary for various diagnostic and research applications. This paper presents acoustic focusing in dense media containing iodixanol to purify peripheral blood mononuclear cells (PBMCs) from whole blood in a label-free and flow-through format. If the blood is laminated or mixed with iodixanol solutions while passing through the resonant microchannel, all the components (fluids and cells) rearrange according to their acoustic impedances. Red blood cells (RBCs) have higher effective acoustic impedance than PBMCs. Therefore, they relocate to the pressure node despite the dense medium, while PBMCs stay near the channel walls due to their negative contrast factor relative to their surrounding medium. By modifying the medium and thus tuning the contrast factor of the cells, we enriched PBMCs relative to RBCs by a factor of 3600 to 11,000 and with a separation efficiency of 85%. That level of RBC depletion is higher than most other microfluidic methods and similar to that of density gradient centrifugation. The current acoustophoretic chip runs up to 20 µl/min undiluted whole blood and can be integrated with downstream analysis.

Biomolecular Interaction Analysis Quantification with a Low-Volume Microfluidic Chip and Particle Diffusometry.

Microfluidic techniques are widely applied in biomolecular analysis and disease diagnostic assays. While the volume of the sample that is directly used in such assays is often only femto-to microliters, the "dead volume" of solutions supplied in syringes and tubing can be much larger, even up to milliliters, increasing overall reagent use and making analysis significantly more expensive. To reduce the difficulty and cost, we designed a new chip using a low volume solution for analysis and applied it to obtain real-time data for protein-protein interaction measurements. The chip takes advantage of air/aqueous two-phase droplet flow, on-chip rapid mixing within milliseconds, and a droplet capture method, that ultimately requires only 2 µL of reagent solution. The interaction is analyzed by particle diffusometry, a nonintrusive and precise optical detection method to analyze the properties of microparticle diffusion in solution. Herein, we demonstrate on-chip characterization of human immunodeficiency virus p24 antibody-antigen protein binding kinetics imaged via fluorescence microscopy and analyzed by PD. The measured kon and koff are 1 × 106 M-1 s-1 and 3.3 × 10-4 s-1, respectively, and agree with independent measurement via biolayer interferometry and previously calculated p24-antibody binding kinetics. This new microfluidic chip and the protein-protein interaction analysis method can also be applied in other fields that require low-volume solutions to perform accurate measurement, analysis, and detection.

A novel electrophoretic assisted hydrophobic microdevice for enhancing blood cell sorting: design and numerical simulation.

Microfluidic technology has great advantages in the precise manipulation of micro-nano particles, and the hybrid microfluidic separation technology has attracted much attention due to the advantages of both active and passive separation technology at the same time. In this paper, the hydrophoresis sorting technique is combined with the dielectrophoresis technique, and a dielectrophoresis-assisted hydrophoresis microdevice is studied to separate blood cells. By using the dielectrophoresis force to change the suspension position of the cells in the channel, the scope of the hydrophoresis device for sorting particles is expanded. At the same time, the effects of microchannel width, fluid velocity, and electrode voltage on cell sorting were discussed, and the cell separation process was simulated. This work has laid a certain theoretical foundation for the rapid diagnosis of diseases in practical applications.

Next generation microfluidics: fulfilling the promise of lab-on-a-chip technologies.

Microfluidic lab-on-a-chip technologies enable the analysis and manipulation of small fluid volumes and particles at small scales and the control of fluid flow and transport processes at the microscale, leading to the development of new methods to address a broad range of scientific and medical challenges. Microfluidic and lab-on-a-chip technologies have made a noteworthy impact in basic, preclinical, and clinical research, especially in hematology and vascular biology due to the inherent ability of microfluidics to mimic physiologic flow conditions in blood vessels and capillaries. With the potential to significantly impact translational research and clinical diagnostics, technical issues and incentive mismatches have stymied microfluidics from fulfilling this promise. We describe how accessibility, usability, and manufacturability of microfluidic technologies should be improved and how a shift in mindset and incentives within the field is also needed to address these issues. In this report, we discuss the state of the microfluidic field regarding current limitations and propose future directions and new approaches for the field to advance microfluidic technologies closer to translation and clinical use. While our report focuses on using blood as the prototypical biofluid sample, the proposed ideas and research directions can be extrapolated to other areas of hematology, oncology, biology, and medicine.

Multiphysics analytical and numerical studies of biomolecule preconcentration utilizing ion concentration polarization: a case study of convergent microchannels.

A convergent sector in microfluidic devices utilizing ion concentration polarization (ICP) can help increase the preconcentration rate and the concentration enhancement factor (CEF) of biomolecules. In this work, we present a detailed study of the nozzle-like-squeeze effect of a convergent channel on the preconcentration of biomolecules. By numerically solving coupled Nernst-Planck-Poisson-Navier-Stokes governing equations for the 2D channel model, we report the first study on the critical width of a convergent region in the channel to retain the advantage of the nozzle-like-squeeze effect in speeding up preconcentration and augmenting CEF. In addition, we investigated the impact of the location and the dimensions of a convergent sector on the mechanism of biomolecule preconcentration. The location of an ion-selective membrane was also determined to ensure that biomolecules are focused on the convergent region of the channel. Moreover, we introduce analytical studies to compare and verify simulation findings. Specifically, the formulas for the critical dimensions of a convergent channel, location of a preconcentrated biomolecule plug, and position of an ion-selective membrane are presented. Furthermore, important working parameters, including electric potentials and hydrostatic pressures, were examined to scrutinize their effect on convergent concentrators. These detailed analytical solutions and numerical simulation results are consistent with experimental observations, providing deep insights into the ICP phenomenon and the preconcentration mechanism of biomolecules in convergent microfluidic concentration devices.

Dynamic video recognition for cell-encapsulating microfluidic droplets.

Droplet microfluidics is a highly sensitive and high-throughput technology extensively utilized in biomedical applications, such as single-cell sequencing and cell screening. However, its performance is highly influenced by the droplet size and single-cell encapsulation rate (following random distribution), thereby creating an urgent need for quality control. Machine learning has the potential to revolutionize droplet microfluidics, but it requires tedious pixel-level annotation for network training. This paper investigates the application software of the weakly supervised cell-counting network (WSCApp) for video recognition of microdroplets. We demonstrated its real-time performance in video processing of microfluidic droplets and further identified the locations of droplets and encapsulated cells. We verified our methods on droplets encapsulating six types of cells/beads, which were collected from various microfluidic structures. Quantitative experimental results showed that our approach can not only accurately distinguish droplet encapsulations (micro-F1 score > 0.94), but also locate each cell without any supervised location information. Furthermore, fine-tuning transfer learning on the pre-trained model also significantly reduced (>80%) annotation. This software provides a user-friendly and assistive annotation platform for the quantitative assessment of cell-encapsulating microfluidic droplets.

SeParate: multiway fluorescence-activated droplet sorting based on integration of serial and parallel triaging concepts.

Fluorescence-activated droplet sorting (FADS) has emerged as a versatile high-throughput sorting tool that is, unlike most fluorescence-activated cell sorting (FACS) platforms, capable of sorting droplet-compartmentalized cells, cell secretions, entire enzymatic reactions and more. Recently, multiplex FADS platforms have been developed for the sorting of multi-fluorophore populations towards different outlets in addition to the standard, more commonly used, 2-way FADS platform. These multiplex FADS platforms consist of either multiple 2-way junctions one after the other (i.e. serial sorters) or of one junction sorting droplets in more than 2 outlets (i.e. parallel sorters). In this work, we present SeParate, a novel platform based on integrating s̲e̲rial and p̲a̲r̲allel sorting principles for accura̲t̲e̲ multiplex droplet sorting that is able to mitigate limitations of current multiplex sorters. We show the SeParate platform and its capability in highly accurate 4-way sorting of a multi-fluorophore population into four subpopulations with the potential to expand to more. More specifically, the SeParate platform was thoroughly validated using mixed populations of fluorescent beads and picoinjected droplets, yielding sorting accuracies up to 100% and 99.9%, respectively. Finally, transfected HEK-293T cells were sorted employing two different optical setups, resulting in an accuracy up to 99.5%. SeParate's high accuracy for a diverse set of samples, including highly variable biological specimens, together with its scalability beyond the demonstrated 4-way sorting, warrants a broad applicability for multi-fluorophore studies in life sciences, environmental sciences and others.

Ultrasimple size encoded microfluidic chip for rapid simultaneous multiplex detection of DNA sequences.

Simultaneous multiplexed analysis can provide comprehensive information for disease diagnosis. However, the current multiplex methods rely on sophisticated barcode technology, which hinders its wider application. In this study, an ultrasimple size encoding method is proposed for multiplex detection using a wedge-shaped microfluidic chip. Driving by negative pressure, microparticles are naturally arranged in distinct stripes based on their sizes within the chip. This size encoding method demonstrates a high level of precision, allowing for accuracy in distinguishing 3-5 sizes of microparticles with a remarkable accuracy rate of up to 99%, even the microparticles with a size difference as small as 0.5 µm. The entire size encoding process is completed in less than 5 min, making it ultrasimple, reliable, and easy to operate. To evaluate the function of this size encoding microfluidic chip, three commonly co-infectious viruses' nucleic acid sequences (including complementary DNA sequences of HIV and HCV, and DNA sequence of HBV) are employed for multiplex detection. Results indicate that all three DNA sequences can be sensitively detected without any cross-interference. This size-encoding microfluidic chip-based multiplex detection method is simple, rapid, and high-resolution, its successful application in serum samples renders it highly promising for potential clinical promotion.

Open microfluidics: droplet microarrays as next generation multiwell plates for high throughput screening.

Multiwell plates are prominent in the biological and chemical sciences; however, they face limitations in terms of throughput and deployment in emerging bioengineering fields. Droplet microarrays, as an open microfluidic technology, organise tiny droplets typically in the order of thousands, on an accessible plate. In this perspective, we summarise current approaches for generating droplets, fluid handling on them, and analysis within droplet microarrays. By enabling unique plate engineering opportunities, demonstrating the necessary experimental procedures required for manipulating and interacting with biological cells, and integrating with label-free analytical techniques, droplet microarrays can be deployed across a more extensive experimental domain than what is currently covered by multiwell plates. Droplet microarrays thus offer a solution to the bottlenecks associated with multiwell plates, particularly in the areas of biological cultivation and high-throughput compound screening.

Metal-enhanced fluorescence biosensor integrated in capillary flow-driven microfluidic cartridge for highly sensitive immunoassays.

Point-of-care testing (POCT) for low-concentration protein biomarkers remains challenging due to limitations in biosensor sensitivity and platform integration. This study addresses this gap by presenting a novel approach that integrates a metal-enhanced fluorescence (MEF) biosensor within a capillary flow-driven microfluidic cartridge (CFMC) for the ultrasensitive detection of the Parkinson's disease biomarker, aminoacyl-tRNA synthetase complex interacting multi-functional protein 2 (AIMP-2). Crucial point to this approach is the orientation-controlled immobilization of capture antibody on a nanodimple-structured MEF substrate within the CFMC. This strategy significantly enhances fluorescence signals without quenching, enabling accurate quantification of low-concentration AIMP-2 using a simple digital fluorescence microscope with a light-emitting diode excitation source and a digital camera. The resulting platform exhibits exceptional sensitivity, achieving a limit of detection in the pg/mL range for AIMP-2 in human serum. Additionally, the CFMC design incorporates a capillary-driven passive sample transport mechanism, eliminating the need for external pumps and further simplifying the detection process. Overall, this work demonstrates the successful integration of MEF biosensing with capillary microfluidics for point-of-care applications.

Magnetic Microbead-Based Herringbone Chip for Sensitive Detection of Human Immunodeficiency Virus.

The microfluidic chip-based nucleic acid detection method significantly improves the sensitivity since it precisely controls the microfluidic flow in microchannels. Nonetheless, significant challenges still exist in improving the detection efficiency to meet the demand for rapid detection of trace substances. This work provides a novel magnetic herringbone (M-HB) structure in a microfluidic chip, and its advantage in rapid and sensitive detection is verified by taking complementary DNA (cDNA) sequences of human immunodeficiency virus (HIV) detection as an example. The M-HB structure is designed based on controlling the magnetic field distribution in the micrometer scale and is formed by accumulation of magnetic microbeads (MMBs). Hence, M-HB is similar to a nanopore microstructure, which has a higher contact area and probe density. All of the above is conducive to improving sensitivity in microfluidic chips. The M-HB chip is stable and easy to form, which can linearly detect cDNA sequences of HIV quantitatively ranging from 1 to 20 nM with a detection limit of 0.073 nM. Compared to the traditional herringbone structure, this structure is easier to form and release by controlling the magnetic field, which is flexible and helps in further study. Results show that this chip can sensitively detect the cDNA sequences of HIV in blood samples, demonstrating that it is a powerful platform to rapidly and sensitively detect multiple nucleic acid-related viruses of infectious diseases.

Trends and challenges in microfluidic methods for protein manipulation-A review.

Proteins are important molecules involved in an immensely large number of biological processes. Being capable of manipulating proteins is critical for developing reliable and affordable techniques to analyze and/or detect them. Such techniques would enable the production of therapeutic agents for the treatment of diseases or other biotechnological applications (e.g., bioreactors or biocatalysis). Microfluidic technology represents a potential solution to protein manipulation challenges because of the diverse phenomena that can be exploited to achieve micro- and nanoparticle manipulation. In this review, we discuss recent contributions made in the field of protein manipulation in microfluidic systems using different physicochemical principles and techniques, some of which are miniaturized versions of already established macro-scale techniques.

Microfluidic Platforms for Real-Time In Situ Monitoring of Biomarkers for Cellular Processes.

Cellular processes are mechanisms carried out at the cellular level that are aimed at guaranteeing the stability of the organism they comprise. The investigation of cellular processes is key to understanding cell fate, understanding pathogenic mechanisms, and developing new therapeutic technologies. Microfluidic platforms are thought to be the most powerful tools among all methodologies for investigating cellular processes because they can integrate almost all types of the existing intracellular and extracellular biomarker-sensing methods and observation approaches for cell behavior, combined with precisely controlled cell culture, manipulation, stimulation, and analysis. Most importantly, microfluidic platforms can realize real-time in situ detection of secreted proteins, exosomes, and other biomarkers produced during cell physiological processes, thereby providing the possibility to draw the whole picture for a cellular process. Owing to their advantages of high throughput, low sample consumption, and precise cell control, microfluidic platforms with real-time in situ monitoring characteristics are widely being used in cell analysis, disease diagnosis, pharmaceutical research, and biological production. This review focuses on the basic concepts, recent progress, and application prospects of microfluidic platforms for real-time in situ monitoring of biomarkers in cellular processes.

Facile prepared microfluidic chip for multiplexed digital RT-qPCR test.

The chip-based digital polymerase chain reaction (PCR) is an indispensable technique for amplifying and quantifying nucleic acids, which has been widely employed in molecular diagnostics at both fundamental and clinical levels. However, the previous designs have yet to achieve widespread application due to limitations in complex chip fabrication, pretreatment procedures, special surface properties, and low throughput. This study presents a facile digital microfluidic chip driven by centrifugal force for digital PCR analysis. Interestingly, regardless of the hydrophilicity or hydrophobicity of the inner chip surface, an efficient digitization process can be achieved. PCR reagents introduced into the inlet can be allocated to 9600 microchambers and subsequently isolated by the immiscible phase (silicone oil). The centrifugal priming approach offers a facile means to achieve high-throughput analysis. The design was further employed for the quantification of nucleic acids using digital PCR. The calculated result exhibited a strong correlation with the measured value at the concentrations from 1 copy/µL to 1000 copies/µL (R2  = 0.99). Additionally, the chip also allowed digital multiplexed analysis, thereby indicating its potential for multi-target detection applications.

A Cascaded Phase-Transfer Microfluidic Chip with Magnetic Probe for High-Activity Sorting, Purification, Release, and Detection of Circulating Tumor Cells.

Microfluidic chips have emerged as a promising tool for sorting and enriching circulating tumor cells (CTCs) in blood, while the efficacy and purity of CTC sorting greatly depend on chip design. Herein, a novel cascaded phase-transfer microfluidic chip was developed for high-efficiency sorting, purification, release, and detection of MCF-7 cells (as a model CTC) in blood samples. MCF-7 cells were specifically captured by EpCAM aptamer-modified magnetic beads and then introduced into the designed cascaded phase-transfer microfluidic chip that consisted of three functional regions (sorting, purification, and release zone). In the sorting zone, the MCF-7 cells moved toward the inner wall of the channel and entered the purification zone for primary separation from white blood cells; in the purification zone, the MCF-7 cells were transferred to the phosphate-buffered saline flow under the interaction of Dean forces and central magnetic force, achieving high purification of MCF-7 cells from blood samples; in the release zone, MCF-7 cells were further transferred into the nuclease solution and fixed in groove by the strong magnetic force and hydrodynamic force, and the continuously flowing nuclease solution cleaved the aptamer on the trapped MCF-7 cells, causing gentle release of MCF-7 cells for subsequent inductively coupled plasma mass spectrometry (ICP-MS) detection or further cultivation. By measurement of the endogenous element Zn in the cells using ICP-MS for cell counting, an average cell recovery of 84% for MCF-7 cells was obtained in spiked blood samples. The developed method was applied in the analysis of real blood samples from healthy people and breast cancer patients, and CTCs were successfully detected in all tested patient samples (16/16). Additionally, the removal of the magnetic probes on the cell surface significantly improved cell viability up to 99.3%. Therefore, the developed cascaded phase-transfer microfluidic chip ICP-MS system possessed high integration for CTCs analysis with high cell viability, cell recovery, and purity, showing great advantages in early clinical cancer diagnosis.

Label-free multidimensional bacterial characterization with an ultrawide detectable concentration range by microfluidic impedance cytometry.

Rapid and accurate identification of bacteria is of great importance to public health in various fields, including medical diagnostics, food safety, and environmental monitoring. However, most existing bacterial detection methods have very narrow detectable concentration ranges and limited detection information, which easily leads to wrong diagnosis and treatment. This work presents a novel high-throughput microfluidic electrical impedance-based multidimensional single-bacterium profiling system for ultrawide concentration range detection and accurate differentiation of viability and Gram types of bacteria. The electrical impedance-based microfluidic cytometry is capable of multi-frequency impedance quantification, which allows profiling of the bacteria size, concentration, and membrane impedance as an indicator of bacterial viability and Gram properties in a single flow-through interrogation. It has been demonstrated that this novel impedance cytometry has an ultrawide bacterial counting range (102-108 cells per mL), and exhibits a rapid and accurate discrimination of viability and Gram types of bacteria in a label-free manner. Escherichia coli (E. coli) has been used as an analog species for the accuracy assessment of the electrical impedance-based bacterial detection system in an authentic complex beverage matrix within 24 hours. The impedance-based quantifications of viable bacteria are consistent with those obtained by the classical bacterial colony counting method (R2 = 0.996). This work could pave the way for providing a novel microfluidic cytometry system for rapid and multidimensional bacterial detection in diverse areas.

Squeezed state in the hydrodynamic focusing regime for Escherichia coli bacteria detection.

Flow cytometry is an essential technique in single particle analysis and cell sorting for further downstream diagnosis, exhibiting high-throughput and multiplexing capabilities for many biological and biomedical applications. Although many hydrodynamic focusing-based microfluidic cytometers have been demonstrated with reduced size and cost to adapt to point-of-care settings, the operating conditions are not characterized systematically. This study presents the flow transition process in the hydrodynamic focusing mechanism when the flow rate or the Reynolds number increases. The characteristics of flow fields and mass transport were studied under various operating conditions, including flow rates and microchannel heights. A transition from the squeezed focusing state to the over-squeezed anti-focusing state in the hydrodynamic focusing regime was observed when the Reynolds number increased above 30. Parametric studies illustrated that the focusing width increased with the Reynolds number but decreased with the microchannel height in the over-squeezed state. The microfluidic cytometric analyses using microbeads and E. coli show that the recovery rate was maintained by limiting the Reynolds number to 30. The detailed analysis of the flow transition will provide new insight into microfluidic cytometric analyses with a broad range of applications in food safety, water monitoring and healthcare sectors.

A Deep Reinforcement Learning Approach to Droplet Routing for Erroneous Digital Microfluidic Biochips.

Digital microfluidic biochips (DMFBs), which are used in various fields like DNA analysis, clinical diagnosis, and PCR testing, have made biochemical experiments more compact, efficient, and user-friendly than the previous methods. However, their reliability is often compromised by their inability to adapt to all kinds of errors. Errors in biochips can be categorized into two types: known errors, and unknown errors. Known errors are detectable before the start of the routing process using sensors or cameras. Unknown errors, in contrast, only become apparent during the routing process and remain undetected by sensors or cameras, which can unexpectedly stop the routing process and diminish the reliability of biochips. This paper introduces a deep reinforcement learning-based routing algorithm, designed to manage not only known errors but also unknown errors. Our experiments demonstrated that our algorithm outperformed the previous ones in terms of the success rate of the routing, in the scenarios including both known errors and unknown errors. Additionally, our algorithm contributed to detecting unknown errors during the routing process, identifying the most efficient routing path with a high probability.

Fully Automated Multiple Standard Addition on a Centrifugal Microfluidic System.

We herein describe a novel centrifugal microfluidic system to achieve multiple standard additions, which could minimize the effects of matrix interference and consequently lead to more accurate and reliable measurements of analyte concentrations in complex samples. The system leverages laser-irradiated ferrowax microvalves to automatically control fluid transfer on the disc without the need for external pumps or pressure systems, simplifying the procedures and eliminating the need for manual intervention. The disc incorporates metering chambers with rationally designed varying sizes, which could lead to the formation of six standard addition samples very rapidly in just 2.5 min. The final solutions are designed to contain a target component at gradually increasing concentrations but have an equal final volume containing the same amount of an analyte solution, thereby equalizing the matrix effect that is supposedly caused by the unknown components in the analyte solution. By utilizing this design principle, we were able to successfully quantify a model target component, salivary thiocyanate ions, that could be used as a biomarker for exposure to tobacco smoke. Our centrifugal microfluidic system holds great promise as a powerful analytical tool to achieve fully automated diagnostic microsystems involving a standard addition process.

pH Regulator on Digital Microfluidics with Pico-Dosing Technique.

Real-time pH control on-chip is a crucial factor for cell-based experiments in microfluidics, yet difficult to realize. In this paper, we present a flexible pH regulator on a digital microfluidic (DMF) platform. The pico-dosing technology, which can generate and transfer satellite droplets, is presented to deliver alkali/acid into the sample solution to change the pH value of the sample. An image analysis method based on ImageJ is developed to calculate the delivered volume and an on-chip colorimetric method is proposed to determine the pH value of the sample solution containing the acid-base indicator. The calculated pH values show consistency with the measured ones. Our approach makes the real-time pH control of the on-chip biological experiment more easy to control and flexible.