Most bioparticles, such as red blood cells and bacteria, are non-spherical in shape. However, conventional microfluidic separation devices are designed for spherical particles. This poses a challenge in designing a separation device for non-spherical bioparticles, as the smallest dimension of the bioparticle has to be considered, which increases fabrication challenges and decreases the throughput. If current methods do not take into account the shape of nonspherical bioparticles, the separation will be inefficient. Here, to address this challenge, we present a novel technique for the separation of red blood cells as a non-spherical bioparticle, using a new I-shaped pillar arrays design. It takes the shape into account and induces rotational movements, allowing us to leverage on the largest dimension, which increases its separation size. This technique has been used for 100% separation of red blood cells from blood samples in a focused stream, outperforming the conventional pillar array designs.

Nanoparticles have been widely implemented for healthcare and nanoscience industrial applications. Thus, efficient and effective nanoparticle separation methods are essential for advancement in these fields. However, current technologies for separation, such as ultracentrifugation, electrophoresis, filtration, chromatography, and selective precipitation, are not continuous and require multiple preparation steps and a minimum sample volume. Microfluidics has offered a relatively simple, low-cost, and continuous particle separation approach, and has been well-established for micron-sized particle sorting. Here, we review the recent advances in nanoparticle separation using microfluidic devices, focusing on its techniques, its advantages over conventional methods, and its potential applications, as well as foreseeable challenges in the separation of synthetic nanoparticles and biological molecules, especially DNA, proteins, viruses, and exosomes.

The advent of microfluidics in the 1990s promised a revolution in multiple industries, from healthcare to chemical processing. Deterministic Lateral Displacement (DLD) is a continuous-flow microfluidic particle separation method discovered in 2004 that has been applied successfully and widely to the separation of blood cells, yeast, spores, bacteria, viruses, DNA, droplets, and more. DLD is conceptually simple and can deliver consistent performance over a wide range of flow rates and particle concentrations.Despite wide use and in-depth study, DLD has not yet been fully understood or fully optimised, with different approaches to the same problem yielding varying results. We endeavour here to provide an up-to-date expert opinion on the state-of-art and current fundamental, practical, and commercial challenges as well as experimental and modelling opportunities. Since these challenges and opportunities arise from constraints on hydrodynamics, fabrication and operation at the micro-and nano-scale, we expect this article to serve as a guide for the broader micro-and nanofluidic community to identify and address open questions in the field.

Particle sorting methods in microfluidic platforms are gaining momentum for various biomedical applications. Bioparticles are found in different shapes and sizes. However, conventional separation techniques are mainly designed for separation of spherical particles. Thus, there is a need to develop new methods for effective separation of spherical and non-spherical bioparticles for various applications. Deterministic lateral displacement (DLD) microfluidic methods have become popular for high separation resolution, simplicity, and predictability. However, shape sorting in the DLD separation methods is not well researched. Recently, we explored this area and found that pillar shapes in DLD significantly affect bioparticle separation. In this work, we designed a group of different pillar shapes with protrusions and groove structures with the hypothesis that pillar protrusions will induce particle rotation while pillar grooves will confine the particle rotational movement in a directed path for effective separation in a DLD pillar array. Using combinations of protrusions and grooves, 3-dimensional spherical particles, 2-dimensional planar disc-shaped red blood cells and 1-dimensional rod-shaped bacteria were separated and two interesting phenomena were observed. Firstly, the arrangement of pillar protrusions and grooves induces inertial movements, enhancing the separation of spherical particles. Secondly, non-spherical particles experience dominant rotational movements due to the protrusions and grooves which help in changing their orientations. This gives an opportunity to perform efficient separation based on the desired orientation (the longest dimension of the particles) by restricting or containing their movement within a specific DLD path.

Nanoparticles exhibit size-dependent properties which make size-selective purification of proteins, DNA or synthetic nanoparticles essential for bio-analytics, clinical medicine, nano-plasmonics and nano-material sciences. Current purification methods of centrifugation, column chromatography and continuous-flow techniques suffer from particle aggregation, multi-stage process, complex setups and necessary nanofabrication. These increase process costs and time, reduce efficiency and limit dynamic range. Here, we achieve an unprecedented real-time nanoparticle separation (51-1500 nm) using a large-pore (2 μm) deterministic lateral displacement (DLD) device. No external force fields or nanofabrication are required. Instead, we investigated innate long-range electrostatic influences on nanoparticles within a fluid medium at different NaCl ionic concentrations. In this study we account for the electrostatic forces beyond Debye length and showed that they cannot be assumed as negligible especially for precise nanoparticle separation methods such as DLD. Our findings have enabled us to develop a model to simultaneously quantify and modulate the electrostatic force interactions between nanoparticle and micropore. By simply controlling buffer solutions, we achieve dynamic nanoparticle size separation on a single device with a rapid response time (<20 s) and an enlarged dynamic range (>1200%), outperforming standard benchtop centrifuge systems. This novel method and model combines device simplicity, isolation precision and dynamic flexibility, opening opportunities for high-throughput applications in nano-separation for industrial and biological applications.

Deterministic lateral displacement (DLD) method for particle separation in microfluidic devices has been extensively used for particle separation in recent years due to its high resolution and robust separation. DLD has shown versatility for a wide spectrum of applications for sorting of micro particles such as parasites, blood cells to bacteria and DNA. DLD model is designed for spherical particles and efficient separation of blood cells is challenging due to non-uniform shape and size. Moreover, separation in sub-micron regime requires the gap size of DLD systems to be reduced which exponentially increases the device resistance, resulting in greatly reduced throughput. This paper shows how simple application of asymmetrical Deterministic lateral displacement (DLD) method for particle separation in microfluidic devices has been extensively used for particle separation in recent years since it was first published by Huang et al.1,2 Due to its high resolution and robust separation, DLD has shown versatility for a wide spectrum of applications for sorting of microparticles such as parasites 3 , blood cells [4][5][6][7] , circulating tumor cells 8,9 , bacteria 7,10 , spores 11 , and more recently, nanoparticle separation 12 and DNA isolation 13,14 . While the DLD empirical model is well established in current research, its scope is restricted to spherical particles, cylindrical pillars and uniform gap-size across all adjacent pillars 15 . Thus, it has been challenging to separate non-spherical particles such as red blood cells (RBCs), bacteria and DNA. Many groups have worked on changing pillar shapes and device gaps to effectively separate these particles 16,17 . However, this will greatly increase fabrication complexity and restrict throughput by reducing gap-sizes in a DLD pillar array.The DLD method uses rhombic or rotated square pillar arrays to redirect fluid laminar flow streams and each array arrangement would have a distinctive critical separating diameter. Particle larger than the critical cut-off diameter (D c ) will be displaced laterally from its flow from the sample streamline while smaller particles flow unhindered in the pillar array by flowing within the fluid streamlines. Based on current DLD empirical model, DLD separation cut-off size and resolution depends on the array rotation angle or slant and lateral gap-size between pillars 6 : ε = .. D g 1 4(1) c 0 48

Disease diagnostics requires detection and quantification of nano-sized bioparticles including DNA, proteins, viruses, and exosomes. Here, a fluorescent label-free method for sensitive detection of bioparticles is explored using a pillar array with micrometer-sized features in a deterministic lateral displacement (DLD) device. The method relies on measuring changes in size and/or electrostatic charges of 1 µm polymer beads due to the capture of target bioparticles on the surface. These changes can be sensitively detected through the lateral displacement of the beads in the DLD array, wherein the lateral shifts in the output translates to a quantitative measurement of bioparticles bound to the bead. The detection of albumin protein and nano-sized polymer vesicles with a concentration as low as 10 ng mL−1 (150 pM) and 3.75 μg mL−1, respectively, is demonstrated. This label-free method holds potential for point-of-care diagnostics, as it is low-cost, fast, sensitive, and only requires a standard laboratory microscope for detection.

advances in disease diagnostics, the main culprit for disease manifestation, severity, and death is the hyper-aggressive host immune response in most instances. [1-3] In the example of severe COVID-19 infection, the leading cause of death is sepsis (dysregulated immune response) while existing risk stratification methods based on age and co-morbidity remain challenging and imprecise. [4,5] The status of the patients' immune response can quickly change in a matter of minutes, therefore assays that are able to rapidly inform on the state of the immune system are vital in early triage among patients with acute infection, as well as prediction of downstream deterioration of disease. [6,7] This enables delivery of appropriate medical response, particularly in the emergency department (ED), for timely intervention before immune dysregulation becomes clinically evident and requiring admission to the intensive care unit (ICU). Unlike patients in the ICU who almost always have clear clinical manifestations of disease severity and organ dysfunction (e.g., low blood pressure, decreased oxygenation, jaundice, low urine output), those in the ED frequently show non-specific symptoms and signs, which pose a challenge for physicians to assess the presence of infection and possibility of deterioration into organ dysfunction. Disease manifestation and severity from acute infections are often due to hyper-aggressive host immune responses which change within minutes. Current methods for early diagnosis of infections focus on detecting low abundance pathogens, which are time-consuming, of low sensitivity, and do not reflect the severity of the pathophysiology appropriately. The approach here focuses on profiling the rapidly changing host inflammatory response, which in its over-exuberant state, leads to sepsis and death. A 15-min labelfree immune profiling assay from 20 µL of unprocessed blood using unconventional L and Inverse-L shaped pillars of deterministic lateral displacement microfluidic technology is developed. The hydrodynamic interactions of deformable immune cells enable simultaneous sorting and immune response profiling in whole blood. Preliminary clinical study of 85 donors in emergency department with a spectrum of immune response states from healthy to severe inflammatory response shows correlation with biophysical markers of immune cell size, deformability, distribution, and cell counts. The speed of patient stratification demonstrated here has promising impact in deployable point-of-care systems for acute infections triage, risk management, and resource allocation at emergency departments, where clinical manifestation of infection severity may not be clinically evident as compared to inpatients in the wards or intensive care units.