Biological particle separation techniques based on microfluidics

Biological particle separation has wide applications in medical diagnosis, bioengineering, and various other domains. Traditional methods, such as filtration, density gradient centrifugation, and size exclusion chromatography, face many challenges, including low separation resolution, low purity, and the inability to be seamlessly integrated into continuous processes. The development of microfluidics has paved the way for efficient and precise biological particle separation. Microfluidic chip‐based methods can generally be performed continuously and automatically, and microfluidic chips can integrate multilevel operations, including mixing, separation, detection, and so forth, thereby achieving continuous processing of particles at various levels. This review comprehensively investigates biological particle separation techniques based on microfluidic chips. According to the different sources of force effect on the particles during the separation process, they can be divided into active separation, passive separation, and affinity separation. We introduce the principles and device design of these methods respectively, and compare their advantages and disadvantages. For the introduction of each method, we used the most classic and latest research cases as much as possible. Additionally, we discussed the differences between experimental standard particles and biological particles. Finally, we summarized the current limitations and challenges of existing microfluidic separation techniques, while exploring future trends and prospects.

Biological particles such as cells, 1 bacteria, 2 viruses, 3 extracellular vesicle, 4,5 organelles, 6 and so forth play crucial roles in clinical diagnosis, biosynthesis, drug delivery, and microbiological control. 7These biological particles are often dispersed within complex mixtures.The separation and enrichment of target biological particles from large volume samples are crucial for these biological and chemical applications. 8,9For example, circulating tumor cells are cancer cells that enter the bloodstream from the primary cancer lesion in cancer patients. 10The number of circulating tumor cells in the blood can be utilized for early cancer diagnosis and postsurgery treatment assessment.But, even in patients with advanced cancer, the number of circulating tumor cells in their blood is rare, typically around 100 per milliliter.Correspondingly, there are approximately 5 million blood cells per milliliter of blood.The presence of a large number of blood cells can seriously interfere with the detection of circulating tumor cells.Therefore, separating and enriching circulating tumor cells from blood has been a hot research topic. 11raditional biological particle separation methods are mainly centrifugation 12 and filtration. 13For example, centrifugation is employed to separate blood components, while filtration is utilized to collect microorganisms in the culture medium.Traditional separation methods exhibit stable performance and wide applicability, and the required equipment is usually simple and readily available.But these methods have obvious limitations, such as low separation resolution, low purity and long time consumption.In particular, these traditional methods typically rely on the physical properties of particles, such as size and density, which limits their usability when dealing with particles that exhibit similar properties.These bottlenecks are challenging to overcome through improvements in traditional methods, prompting researchers to shift their focus toward the development of new biological particle separation methods. 14icrofluidics is a technology that manipulates fluids in the scale range from submicron to hundreds of microns. 15Microfluidic technology is implemented through a microfluidic chip, also known as lab-on-a-chip.Microfluidic channels, microvalves, and micropumps can be created on the chip through micro-nano processing to achieve highly precise microfluid control. 16The micrometer-scale characteristics of microfluidics provide a resolution far superior to traditional separation methods in manipulating and separating biological particles.According to the different sources of force effect on the particles during the separation process, they can be divided into active separation, passive separation, and affinity separation. 17Active separation mainly relies on external force fields to drive the particles.3][34][35] In addition, the gravity field 36,37 is also used as a driving force or auxiliary force for particle separation processes.Passive separation does not require any external force field and relies only on the specially designed channel structure and the fluid mechanics principles corresponding to the channel structure.Fundamentally, the main forces experienced by particles in the passive separation method include shear gradient-induced force, wall-induced lift force, centrifugal force, collision force, and inertial force.These five forces can give rise to different resultant forces, such as the flow-induced inertial lift force and Dean force in microchannels with different structures. 380][61][62][63][64] Thus, the separation performance of affinity separation mainly depends on the specificity and binding strength of the affinity pair.The comparison of different separation methods is summarized in Table 1.
This review aims to provide a comprehensive introduction of microfluidic technology for biological particle separation, not only from the perspective of developers, but also from the perspective of users.Therefore, individuals with specific needs can choose the most suitable separation method according to their requirements.Thus, we will pay special attention to presenting classical and concise application cases and also providing the latest technological advancements.This review elucidates the principles, device design, advantages, and disadvantages of these methods based on the classification of active separation, passive separation, and affinity separation.Additionally, we discussed the differences in properties such as density, uniformity, deformability, and adhesion between experimental standard particles and biological particles.Finally, we summarized the current limitations and challenges of existing microfluidic separation techniques, while exploring future trends and prospects.

| ACTIVE SEPARATION METHODS
Active separation relies on external force fields such as acoustic, electric, magnetic, optical, and thermal fields to control particles within microfluidic channels for separation.Most external forces can be applied directly to the target particles rather than indirectly controlling them by influencing fluid flow.Thus, active separation techniques are highly flexible and can precisely control particles as needed.7][98] In the following section, we will introduce the principles, applicable scenarios, and the current research status of each active separation method.

| Acoustic separation
][107][108] BAW can manipulate particles within a volume, allowing for a wider range of manipulation in threedimensional space, but the accuracy may be relatively low.Surface acoustic wave can be employed to precisely manipulate particles in microfluidic channels, often utilized for the manipulation of nanoscale particles.Despite the differences in their sound wave propagation modes and applicable scenarios, the core components of the systems are all acoustic wave transducers.Acoustic wave transducers can convert electrical signals into sound signals.Bulk acoustic wave transducers are typically made of piezoelectric ceramics, while SAW transducers are typically composed of interdigital electrodes. 100coustic separation has relatively matured in the processing of micron-sized particles, especially in the separation of circulating tumor cells.The work presented in Figure 1A is among the initial successful cases of separating circulating tumor cells from blood using the acoustic method. 109The researchers spiked three types of tumor cells into blood that had lysed red blood cells.For tumor cells fixed with paraformaldehyde, the average recovery rate is approximately 95%, and the average purity is approximately 98%.For non-fixed viable cells, the average recovery rate for tumor cells is approximately 80%, with an average purity of approximately 90%.This work also found that the activity and proliferation ability of tumor cells did not experience significant loss after acoustic separation.This early work used blood in which red blood cells had been lysed.In the latest research (Figure 1B), it has been possible to separate tumor cells from blood without any pre-treatment. 110In this work, researchers spiked active human breast cancer tumor cells directly into human whole blood.After processing with the acoustic device, the final recovery rate was approximately 98%, the purity was about 93%, and even after 72 h of culture, the cell activity still reached 97%.These two typical studies demonstrate that acoustic methods are highly effective in cell separation and gentle enough to avoid affecting cell viability.Separating exosomes is another important application of AP.Researchers have developed an acoustic device (Figure 1C) capable of directly separating exosomes from undiluted whole blood samples. 65The device consists of a two-stage separation system.That is, the first stage of the system is used to remove blood cells from the blood.This process can remove more than 99.999% of blood cells.The second stage of the system can remove non-exosome biological particles in plasma, mainly extracellular vesicles with a particle size greater than 150 μm.The researchers finally separated exosomes from whole blood with a purity of approximately 98%.This research team further developed a higher-precision small extracellular vesicle separation technique (Figure 1D). 20They have pushed the limit of separation resolution to approximately 50 nm in this work.To the best of our knowledge, this is the highest resolution record of all acoustic separation methods so far.In summary, acoustic separation has been widely employed for separating biological particles, including exosomes, 20,101 bacteria, 66,111 blood cells, 112,113 circulating tumor cells, 114,115 and so forth, 116 with sizes ranging from hundreds of nanometers to tens of micrometers.This method is entirely non-contact, highly biocompatible, and versatile.In particular, the sound field properties can be adjusted at any time through the acoustic wave transducer, and the sound field response is F I G U R E 1 Microfluidic-based acoustic separation techniques for biological particles.(A) Separating tumor cells from blood with lysed red blood cells.Reproduced with permission. 109Copyright 2012, American Chemical Society.(B) Separating tumor cells from untreated whole blood.Reproduced with permission. 110Copyright 2023, Elsevier.(C) Separating exosomes from whole blood.Reproduced with permission. 65Copyright 2017, PNAS.(D) Separating exosomes and small exosomes from small extracellular vesicles.Reproduced with permission. 20Copyright 2022, AAAS.
instantaneous.The high degree of controllability makes it easier for users to handle various separation scenarios.However, the intensity of the acoustic radiation force generated by the acoustic field is limited.While this limitation is beneficial for maintaining the activity of biological particles, it also results in lower processing efficiency.Thus, current acoustic separation devices generally suffer from the disadvantage of low throughput.
In addition, the primary obstacle currently impeding the development of acoustic separation technology in the field of biomedical engineering is the requirement of specialized instruments.Equipment such as acoustic wave transducers, function generators, and power amplifiers is not commonly used in biomedical laboratories.Constructing a complete acoustic separation system requires a wealth of professional knowledge and practical experience.Therefore, we believe that the commercial development of integrated equipment will significantly enhance the application of acoustic separation technology in biomedicine.

| Electrical separation
Microfluidic-based electrical separation methods mainly include electrophoresis (EP) and dielectrophoresis (DEP).Electrophoresis is the phenomenon of directional motion of particles in a spatially uniform electric field.Since the electric field in space is uniform, particles must be charged to be driven.Charged particles will migrate in the direction of an electric field that is opposite to their charge.Dielectrophoresis is the phenomenon of directional movement of particles in a spatially non-uniform electric field.In a non-uniform electric field, particles will be polarized and become charged.So, the particles themselves can have no net electric charge.The migration direction of particles is determined by both the dielectric constant of the particle itself and the dielectric constant of the medium.If the dielectric constant of the particles is larger, they migrate toward the positive electrode of the non-uniform electric field.Conversely, they migrate toward the negative electrode if the dielectric constant is smaller.These two situations are called positive DEP and negative DEP, respectively.They can all be used for separating biological micro/nanoparticles.But in general, DEP has higher flexibility and controllability than EP, and it is also the most commonly used electrical method based on microfluidic chips.
Electrophoresis is most commonly used for the separation of biomolecules.A simple EP device only requires a set of parallel electrodes on both sides of the channel.So, EP can be easily combined with other technologies.The work illustrated in Figure 2A integrates EP technology into droplet microfluidic devices. 68The researchers employed droplet microfluidics to generate droplets containing biomolecules such as lambda DNA.Subsequently, EP facilitated the migration of biomolecules toward one side of the droplets.Finally, at the Y-shaped separation port, the droplet divides into sub-droplets, with one containing many biomolecules and another containing little or no biomolecules.In this work, the biomolecule enrichment rate was as high as 95% when the droplet generation rate was 13 drops per second.We believe that this system is highly generalizable, making this work very enlightening.Electrophoresis also has good performance in the separation of micron-scale particles.The interface between liquids of different components can act as a filter.Based on this, the researchers developed an aqueous twophase system EP system (Figure 2B). 69They used EP to facilitate microalgae cell separation by selectively crossing the interface.In this work, the separation of Chlorella sp. and Isochrysis sp. was successfully achieved.Dielectrophoresis is also commonly used for cell separation.Researchers have developed many dielectrophoretic devices for tumor cell separation.Based on the difference in dielectric constant of the tumor cells and blood cells, they can be directed to opposite sides of the channel to achieve separation (Figure 2C). 117Since the diameter of the cell is relatively large, the ratio between the number of charges on the cell surface and the mass of the cell is small, resulting in slow cell migration speed.This means that separation performance is very sensitive to flow rate.Dielectrophoresis is also well-established for the manipulation of nanoparticles.Researchers have developed an exosome separation device based on DEP (Figure 2D), 67 which can capture and separate exosomes within 20 s.This device can capture different subpopulations based on the particle size of exosomes.In this work, the researchers captured exosomes with average particle sizes of 120 and 75 nm, respectively.
All biological particles either have a net charge themselves or can acquire charges through polarization, so the electrical methods have wide applicability.Electrophoresis and DEP do not require labeling of particles, are simple in principle, and have high controllability.Moreover, the magnitude of the electric field force has a linear relationship with the particle size range, rather than the more common cubic relationship.That is to say, the electric field force will not decay rapidly as the particle size decreases.Thus, the electrical methods also have good performance in separating nanoparticles.However, the presence of an electric field will inevitably generate Joule heat.On one hand, the accumulation of heat may potentially damage the activity of biological particles.On the other hand, it could lead to the generation of bubbles in the channel.In addition, the chemical reaction between the electrode and the electrolyte solution will also affect the performance of the device.

| Magnetic separation
The magnetic field separation method on microfluidic chips is generally called magnetophoresis. 95Magnetophoresis refers to the phenomenon in which particles undergo directional movement in a liquid medium under the influence of a magnetic field.Magnetic susceptibility of particles and liquid media is a key parameter in magnetophoresis technology.When the magnetic susceptibility of the particles is greater than that of the liquid medium, positive magnetophoresis occurs, and the particles migrate along the direction of the magnetic field gradient.Otherwise, the particles migrate against the direction of the magnetic field gradient.Since most biological particles are non-magnetic or have very weak magnetic properties, it is necessary to use magnetic beads to label biological particles for indirect manipulation. 118,119uilding a magnetophoretic separation system is not difficult.The simplest and classic structure consists of only a permanent magnet that is used to introduce a magnetic field into the microfluidic channel (Figure 3A). 120The work shown in Figure 3B proposes a magnetophoretic device for separating white blood cells from peripheral blood. 121The device is divided into a lateral separation stage and a vertical separation stage to achieve high recovery rates and high purity of white blood cells, respectively.Similarly, magnetophoresis is often used to isolate circulating tumor cells (Figure 3C). 73n these works, the target cells need to be labeled in advance using magnetic beads.Researchers have also developed magnetic separation methods that do not require pre-labeling in recent years.The work shown in Figure 3D purified circulating tumor cells by capturing white blood cells using magnetic traps. 74This negative separation method does not require labeling circulating tumor cells or exposing them to magnetic fluids that may damage cell activity.Therefore, this is a highly biocompatible separation strategy.
The advantage of magnetophoresis is that it can achieve flexible manipulation of particles by controlling the intensity and direction of the magnetic field.The magnetic field force can be increased by strengthening the magnetic field, thereby reducing the time required for magnetophoresis to achieve rapid separation.An obvious disadvantage of magnetophoresis is the requirement to label the target particles in most cases.Sample preparation is often time-consuming and laborintensive, and the use of magnetic beads may lead to potential damage to biological particles.The elution of magnetic beads from biological particles after the separation process is also cumbersome.These drawbacks limit the universality of magnetophoresis.But this method can be utilized in some specific situations, such as separating particles with similar properties except for magnetic susceptibility.

| Optical separation
Optical separation is commonly known as optical tweezers and optofluidics. 96,122,123Light striking the particles will generate a light scattering force and gradient force.The direction of the light scattering force is consistent with the direction of the light, which can push the particles to move in the direction of light in the liquid medium.The light scattering force is related to the diameter and optical properties of the particles.The direction of the gradient force is consistent with the direction of the intensity gradient of light, which can push particles to follow the movement of light in the liquid medium.It controls the movement of particles just like tweezers, so it is called optical tweezers.The light scattering force and gradient force both have different application ways.
In the method for separating particles based on light scattering force, the laser direction is perpendicular to the direction of particle movement (Figure 4A). 123Under the action of the scattering force, particles move different distances along the direction of laser propagation so separation can be achieved.Because the particles pass through the beam range quickly, they may not have time to be completely separated.Researchers developed an optofluidic platform based on a silicon waveguidepair array to enhance particle separation (Figure 4B). 124Essentially, this method repeatedly irradiates the particle stream with multiple laser beams to extend the deflection time of particles.The researchers claim that their device can theoretically achieve resolutions with an accuracy of 5 nm.Using this optofluidic platform, researchers successfully separated Staphylococcus aureus from a mixture of bacterial nanoparticles.This separation method based on light scattering force is simple to operate, does not require complex optical equipment, and is suitable for simultaneously processing a large number of particles.Optical tweezers, which are separations based on light gradient force, are more F I G U R E 3 Microfluidic-based magnetic separation techniques for biological particles.(A) A simple magnetic separation system.Reproduced with permission. 120Copyright 2017, American Chemical Society.(B) Separation of white blood cells from peripheral blood.Reproduced with permission. 121Copyright 2019, Elsevier.(C) Circulating tumor cell microseparator based on transverse magnetophoresis.Reproduced with permission. 73Copyright 2013, American Chemical Society.(D) Capture white blood cells to achieve negative isolation of circulating tumor cells.Reproduced with permission. 74Copyright 2022, The Royal Society of Chemistry.
suitable for single-cell operations.In the research depicted in Figure 4C, the researchers employed fluorescent excitation light to illuminate the entire microfluidic channel, inducing fluorescence emission from the cells within the channel. 29Different types of cells can be distinguished based on their fluorescence intensity, and optical tweezers are subsequently employed to guide these distinct cells into separate recycling channels.To put it simply, the whole process includes two steps: cell identification based on fluorescence and cell separation based on optical tweezers.Thus, accurate identification of cell types is also important for enhancing separation performance.Researchers have attempted to apply computer image identification techniques to this stage to identify multiple characteristics of cells simultaneously. 126Due to the limitations in force magnitude of traditional optical tweezers, researchers developed an alternative technique known as lightinduced electrokinetic separation, also called optoelectronic tweezers (Figure 4D). 125Optoelectronic tweezers combine the flexibility of optical methods with the sensitivity of electrical methods.This method is currently a major research topic in optical separation.
Optical tweezers offer extremely high resolution, and optical tweezers are non-invasive techniques that do not require any additional labeling.Particles can be easily manipulated in three dimensions using external lasers, offering high flexibility.However, the light scattering force and gradient force are generally weaker than other active methods.Thus, the particle throughput of optofluidic chips is generally not high.In addition, the temperature of the medium will increase after absorbing light, and the biological particles themselves will also absorb light energy and generate heat.Biocompatibility issues limit the application of optofluidics in biological particle separation.
thermophoresis is that thermal gradients induce the convection of liquid media, thereby driving the movement of particles. 128Compared to other active separation methods based on microfluidic chips, thermal methods have been infrequently studied, and consequently, they are seldom encountered in similar reviews.Thermal gradient fields can be generated using electric heating, light-induced heating, and the direct application of hot and cold water.
The study shown in Figure 5A found that only particles of a particular size can be captured for a particular electrothermal current value or a certain thermal gradient. 35his phenomenon can be used to separate particles based on their size.Similarly, this method has also been used for the enrichment of exosomes (Figure 5B). 32,129The separation strategy is achieved by capturing specific particles, much like tweezers, so it is often called thermal tweezers.The work shown in Figure 5C demonstrates another thermophoretic separation strategy. 130Particle separation can be achieved by utilizing different movement speeds of different particles under the same thermal gradient field.In this work, the researchers achieved separation of 0.5 and 1 µm particles.
Thermal separation has been widely used in processing nanoparticles such as exosomes.However, it is not efficient in handling larger particles.Since other active separation methods have been highly developed, developing on-chip thermophoresis is of limited interest among researchers.

| PASSIVE SEPARATION METHODS
Passive separation does not require any external force field and only relies on the specially designed channel structure F I G U R E 5 Microfluidic-based thermal separation techniques for biological particles.(A) Separation of particles based on thermal tweezers.Reproduced with permission. 35Copyright 2018, Elsevier.(B) Enrichment of exosomes based on thermophoresis.Reproduced with permission. 129Copyright 2022, Wiley.(C) Separation of particles based on thermophoresis in an expansion-contraction microchannel.Reproduced with permission. 130Copyright 2019, Elsevier.and the fluid mechanics principles corresponding to the channel structure. 96,131Passive separation often has special requirements for fluid flow, so the flow rate cannot be too fast or too slow.Channels of different sizes in various methods typically correspond to a certain optimal value or range.This also means that there is an upper limit to the particle throughput for a single channel or single device.Additionally, the roughness of the channel in passive separation will affect the fluid flow, thereby affecting the separation performance.In the following section, we will introduce the principles, applicable scenarios, and the current research status of each passive separation method.

| Microfiltration
Microfiltration can be divided into two types, blocked filtration and non-blocked filtration.The principle of blocked filtration is to capture particles with a diameter larger than the filter pores while allowing particles smaller than the filter pores to pass through the filter membrane.In this filtration method, the flow direction of particles is parallel to the normal direction of the filter pores. 76Non-blocking filtration is also called tangential flow filtration, in which the flow direction of particles is perpendicular to the normal direction of the filter holes. 53herefore, although the principle also allows small particles to pass and intercept large particles, large particles can leave the filter pores under the wash of the fluid without causing blockage.
Filtration methods based on microwells are most similar to traditional filtration (Figure 6A).But, the assembly of this type of device is a bit inconvenient.Devices based on micropillars only need to undergo traditional microfluidic chip processing steps (Figure 6B). 75So, micropillar filtration is more common.Both of these methods belong to blocked filtration.The work shown in Figure 6C is a typical non-blocking filtration. 53As illustrated in this work, researchers usually use sheath fluid to enhance filtration.Nonetheless, there is still a risk of clogging with such devices.Researchers have developed a lobe filtration system by mimicking the filter-feeding mechanism of mantas (Figure 6D). 77In this type of device, by carefully designing the distance between filter columns and adjusting specific flow rates, large particles can be induced to jump through the filter holes.Moreover, because the filter pores are larger than the diameter of the biggest particles, there is almost no risk of clogging.However, there are always some large particles that accidentally jump into the filter holes and mix with small particles, limiting the filtration purity.
The advantage of microfiltration is its simple principle, lack of labeling requirements, and the ability to freely control the cut-off size through the pore size of the filter membrane.However, like traditional filtration methods, microfiltration is very prone to clogging.Even nonclogging filtration will gradually become clogged and scaled over time in practical applications.For biological particles without a hard shell such as blood cells, the deformability of cells may result in filter failure and cause active damage due to shearing forces when passing through the filter pores.

| Dean flow separation
The inclusion of curvatures in the spiral channel induces a secondary cross-sectional flow field perpendicular to the primary flow direction, known as Dean flow (Figure 7A). 132Dean flow exerts a Dean force on particles, which leads to the ability of particles in a spiral channel to migrate across the main streamlines by adhering to a secondary vortex.Under the combined influence of the inertial lift force and Dean force, particles of varying sizes correspond to distinct equilibrium positions within the cross-section of the spiral channel.Eventually, larger particles migrated to the inner wall of the channel, while smaller particles migrated to the outer wall of the channel.
Dean flow separation is among the most widely adopted and well-established passive separation methods.The traditional Dean flow separation device is depicted in Figure 7A, and the cross-section of its spiral channel is rectangular. 132Rectangular structures are easily fabricated by conventional photolithography.Rectangular structures can be easily fabricated using conventional photolithography, making this design the most commonly utilized one.Researchers have attempted to enhance separation performance by altering the channel cross-sectional structure, such as using a triangular or trapezoidal cross-section.In spiral microchannels characterized by trapezoidal cross-sections, the unevenness in the trapezoidal shape has an impact on the velocity distribution.This asymmetry leads to the generation of enhanced Dean flow near walls with larger channel depths when contrasted with the flow in rectangular channels.Researchers developed a Dean flow device utilizing a trapezoidal cross-section for plasma separation (Figure 7B). 133At the optimal flow rate of 1.5 mL/min, the slanted spiral channel achieves a rejection ratio nearing 100% for blood samples exhibiting hematocrit values of 0.5% and 1%.Additionally, the researchers assembled a high-throughput system comprising 16 parallel-connected spiral channels, facilitating the rapid separation of plasma and blood cells at a rate of 24 mL/ min.This is a remarkably high speed, and we believe it is adequate for most cases.Additionally, in this study, it is observed that the trapezoidal cross-section of the channel is designed such that the inner wall is higher than the outer wall.There are also structural designs with the outer wall higher than the inner wall, which has a similar effect. 136,137In summary, the strong Dean vortex core is always close to the higher channel wall.The work in Figure 7C demonstrates another strategy to enhance separation. 134The researchers used the double helix structure to change the final equilibrium position of the particles in the channel.In contrast to the single spiral device, the double spiral device exhibits superior focusing capabilities for small blood cells.Although separation devices based on Dean flow do not necessitate the use of sheath fluid, employing sheath liquid can enhance separation performance to obtain higher purity and recovery rate (Figure 7D). 135,138This is because the sheath liquid can reduce the interaction between particles and accelerate the migration of particles.
The significant advantages of the Dean flow separation method, which is characterized by spiral channels, are high throughput and extremely simple operation.It requires almost no other equipment except a pump.It is almost the easiest of all separation methods to try.Although this method is frequently employed for cell separation, its separation resolution is low, thus limiting its capability to separate submicron particles.In the laboratory, this method is often used as a pretreatment step for more precise separation methods.

| Pinched flow fractionation
Pinched flow fractionation (PFF) is a size-based separation method that requires sheath fluid, and its principle is shown in Figure 8A. 43The particle solution and the sheath solution are introduced into the microfluidic channel from two respective inlets and converge at the pinched segment.The flow rate of the sheath fluid is typically much higher than that of the particle solution.In this case, the solution containing particles will be concentrated on the side walls of the pinched segment.When the flow rate of the sheath fluid is high enough, all particles will flow close to the side walls of the channel.Therefore, streamlines corresponding to the centers of particles of different diameters are distinct.The smaller the diameter, the closer the particle centers are to the channel wall, while the larger the diameter, the farther the particle centers are from the channel wall.Thus, particles of different diameters are separated into different streamlines.The Reproduced with permission. 43Copyright 2004, The Royal Society of Chemistry.(B) Extracting viruses from semen employing pinched flow fractionation.Reproduced with permission. 79Copyright 2021, The Royal Society of Chemistry.(C) Inertia-enhanced pinched flow fractionation.Reproduced with permission. 44Copyright 2015, American Chemical Society.(D) Reverse flow enhanced inertia pinched flow fractionation.Reproduced with permission. 45Copyright 2023, American Chemical Society.
separation distance between particles in the pinched segment is small, but in the subsequent abruptly broadened segment, the separation distance can be significantly amplified.This method has been widely used in many fields, such as the removal of viruses from pig semen (Figure 8B). 79In this study, researchers reported that the sperm recovery rate of this method is twice that of other virus separation techniques.
As mentioned previously, pinched flow fractionation requires a very high sheath fluid ratio.This limits the device's particle throughput.As shown in Figure 8C, the researchers exploited flow-induced inertial lift in microchannels to enhance device performance and proposed inertia-enhanced pinched flow fractionation (iPFF). 44,45nertia-enhanced pinched flow fractionation is achieved by simply elongating the pinched segment in the PFF.In the extended pinched segment, the particles will be significantly affected by the flow-induced inertial lift force.Compared with traditional pinched flow fractionation, the separation distance between particles of different diameters is greater.More importantly, at the same sheath flow rate, the particle throughput of this inertia-enhanced pinched flow fractionation method is at least one order of magnitude higher than that of the traditional method.
Based on iPFF, researchers further proposed reverse flow enhanced inertia pinched flow fractionation (RF-iPFF, Figure 8D). 46The change is the establishment of two symmetrical reverse flow channels in the abruptly broadened segment.In this device, the streamlines near the channel wall in the extended pinched segment will enter the reverse flow channel, and the particles corresponding to these streamlines will also enter the reverse flow channel.Compared with iPFF, RF-iPFF consumes less sheath fluid, resulting in a higher particle throughput.Researchers have validated the applicability of the device for biological particles by separating active tumor cells from untreated whole blood.
The advantage of the pinched flow fractionation method is high recovery and purity, while the disadvantage is limited particle throughput and reliance on sheath fluid.This method is suitable for applications with small sample volumes and no stringent requirements for separation speed.

| Deterministic lateral displacement separation
Deterministic lateral displacement is a size-based method for separating particles, which utilizes a unique flow field created by a specially arranged micropillar array (Figure 9A). 139In such devices, the overall arrangement of the micropillar array is tilted to the overall flow direction of the sample solution.All particles will collide (including contactless) with the micropillar during solution flow.Particles smaller than the critical diameter will continue to move in a straight line even after repeated collisions with the micropillars.However, particles larger than the critical diameter will exhibit lateral displacements upon colliding with the micropillars, causing them to move obliquely.The critical diameter can be determined based on the distance between micropillars and the degree of tilt.
The shape and arrangement of the micropillar array directly determine the separation performance.Common micropillar shapes include circles, triangles, squares, and so forth.The arrangement is mostly parallelogram.Researchers developed a hexagonally arranged device for enhanced separation performance (Figure 9B). 140This special arrangement enhances the sorting ability of smallsized particles.Deterministic lateral displacement has been widely used in cell separation.In addition to optimizing the micropillar array, the use of sheath fluid is also an effective way to enhance separation.The sheath fluid can reduce the negative effects of collisions between particles.The work depicted in Figure 9C well separated hemocytes using this method. 80he advantage of Deterministic lateral displacement is that it is simple to operate and suitable for separating particles of various sizes.This method has high resolution and can be used for separating nanoparticles such as exosomes. 81,141However, the precision of the device must also reach the nanometer level for separating nanoparticles, often necessitating electron beam exposure instead of traditional ultraviolet exposure.Thus, this method is most commonly used to separate micron-sized particles.The most obvious disadvantage of deterministic lateral displacement is that the channel is prone to clogging, so the particle throughput of these devices is generally not high.Additionally, this method may not be suitable for cells with strong deformability.

| Contraction-expansion array separation
The distinctive feature of the contraction-expansion array separation chip is the inconsistent cross-sectional width of the microfluidic channel.Visually, it appears wide in certain areas and narrow in others and is characterized by multiple identical cycles of wide and narrow sections.Contraction-expansion array separation primarily leverages the inertial effect of fluid.In microfluidic channels, the inertial effect of the fluid induces the inertial migration of particles and the generation of secondary flows within the fluid itself.Inertial migration is the result of a combination of shear gradient lift and wall lift, while secondary flows exert Dean forces on the particles.The resultant force of these effects guides particles of various sizes to distribute along different streamlines.
The asymmetric serpentine (S-shaped) contractionexpansion array is one of the most common separation structures (Figure 10A). 82Nevertheless, this structure typically requires the determination of multiple parameters, such as curvature and turn width, and researchers often need extensive experience to develop a design that meets their requirements.The rectangular serpentinecurved microchannel depicted in Figure 10B is structurally simpler and easier to design. 142However, it is obvious that the rectangular channel is not completely hydrodynamically designed.Besides snakes and rectangles, triangles are also suitable. 83While most contractionexpansion array separation microchannel structures are asymmetric, symmetric channels can also achieve particle separation (Figure 10D). 143Due to the symmetry of the channels, this type of chip generally contains at least three particle recovery channels: a large particle recovery channel in the middle and symmetrical small particle recovery channels on both sides.The advantage of contraction-expansion array separation is that it has high throughput and does not require sheath fluid.However, due to its low separation resolution, this method is generally limited to handling micron-sized particles.

| AFFINITY SEPARATION METHODS
Affinity separation, also known as immunoaffinity capture, is primarily based on the specific binding between antigens and antibodies or other immune-like interactions.Many biological particles carry antigens on their surfaces, which can only be recognized and bound by specific antibodies.Antibodies can be pre-immobilized on channel surfaces, magnetic beads, and other solidphases.By utilizing the specific binding between antigens and antibodies, biological particles can be indirectly immobilized on the solid-phase surfaces.Affinity separation includes positive separation and negative separation.Positive separation refers to directly capturing target particles and then releasing the target particles.Negative separation refers to capturing non-target particles.Positive separation is suitable for separating rare cells, while negative separation is suitable for further purifying samples containing small numbers of non-target cells.
The key to affinity separation is to ensure full contact between the antigen and antibody.Researchers developed a herringbone chip that can generate microvortex within the channel for greatly enhanced mixing. 84Compared to traditional flat-walled microfluidic devices, microvortexes disrupt the linear traveling pattern of cells in microchannels, causing them to move erratically.This strategy significantly enhances the probability of contact between cells and antibody-coated surfaces.The method of using microvortexes to enhance affinity separation performance has been widely adopted.Researchers developed a wearable system for separating circulating tumor cells in the body. 85The circulating tumor cell capture module of this system also utilized a herringbone-structured chip for achieving rapid capture.
Affinity-based separation methods exhibit high specificity and sensitivity, making them suitable for separating biological particles at very low concentrations.However, nonspecific binding limits the recovery and purity of this method.Affinity-based materials are generally expensive, which may be a consideration, especially in the case of large-scale applications.

BIOPARTICLE SEPARATION TECHNOLOGY
Despite significant progress in bioparticle separation methods based on microfluidic chips over more than 2 decades of development, this technology still requires further enhancement in scalability and commercialization.Researchers in this field generally encounter the following challenges: transitioning from standard particles to biological particles and integrating multiple methods into a cohesive application.

| Challenges from methodology to application
Most biological particles are not readily available.For example, cell culture is time-consuming and laborintensive, so standard particles are often used in methodological studies.In methodological research on microfluidic separation technology, polystyrene particles are frequently employed as substitutes for biological particles, while silica microspheres are occasionally reported.This is because the density of polystyrene (~1 g/cm 3 ) is very similar to that of biological particles such as cells (~1 g/cm 3 ), while the density of silica microspheres is as high as 2.65 g/cm 3 .For particles of the same size, differences in density directly result in variations in inertial force, consequently influencing the mode of movement and migration distance of particles in microchannels.Moreover, the uniformity of standard particles is significantly superior to that of biological particles.This implies that separation methods based on particle size will be greatly influenced.In addition, the deformability of biological particles (mainly cells) affects almost all passive separation methods.The flow of fluid will generate a shear force, which will deform the biological particles.Especially for methods such as microfiltration and deterministic lateral displacement, the deformability of biological particles can invalidate intrinsic chip parameters such as filter pore diameter.Besides, adhesion between standard particles can be eliminated using various chemical modification methods, whereas dispersing bioparticle clusters poses challenges due to the activity of bioparticles.
There are many differences between standard particles and biological particles in properties such as density, uniformity, deformability, and adhesion.These differences pose challenges ranging from methodological to practical applications.

| Challenges from individual- method to hybrid-method integration
Individual methods always have limitations that are difficult to overcome.Integrating different technologies on the same microfluidic chip or connecting multiple microfluidic chips in series can combine the advantages of each method to synergistically overcome the limitations of individual methods. 144However, integrating or coupling multiple physical fields on the same chip is highly challenging, and connecting different chips involves precise control of fluid pressure drops, which is also difficult to achieve.Therefore, research on separating biological particles using hybrid methods is scarce.
Current hybrid microfluidic separation methods can only combine a few techniques, such as affinity separation combined with microfiltration, 60 spiral inertial separation and serpentine inertial separation combined with magnetic separation, 145 spiral inertial separation combined with deterministic lateral displacement, 146 electric field separation combined with magnetic field separation, 147 and electric field separation combined with sound field separation. 148These hybrid methods do enhance separation performance, but in most cases, these attempts focus on further improving the advantages of the original technology rather than fundamentally addressing its disadvantages.The hybrid separation method was tried as early as 10 years ago, but there are still few results so far.This may be because it takes a lot of time to build and debug a hybrid microfluidic separation system, and the engineering challenges are so high that researchers are unwilling to invest.

| DISCUSSION AND PERSPECTIVES
This paper investigated microfluidic techniques for biological particle separation.We classify these techniques into active separation, passive separation, and affinity separation.For active separation, we discussed five common methods based on electric, magnetic, light, acoustic, and thermal fields.For passive separation, we discussed four common methods based on Dean flow, pinch flow, deterministic lateral displacement, and microfiltration.For affinity separation, we discussed two strategies including positive separation and negative separation.For each specific separation method, we introduced its principles, advantages, disadvantages, and classic or easy-to-understand application cases.To facilitate the understanding of readers with different backgrounds, each mentioned method is presented in the form of schematic diagrams, and formulas are avoided as much as possible.We provide corresponding references to help readers obtain further details.
Almost all separation methods are very sensitive to particle concentration, especially for passive separations.This is because passive separation relies on the flow field, and the particles themselves can strongly influence the distribution of the flow field.Similar reasons also influence active separation, especially in light field-based separation methods.Thus, most current devices perform well when handling dilute particle solutions, but the separation performance drops sharply as the particle concentration increases.This means that samples with higher particle concentrations, such as blood, need to be diluted before the separation process.Particle throughput is a critical limitation for microfluidic devices from the laboratory to practical applications.Parallel connecting multiple microfluidic channels or microfluidic devices to achieve simultaneous processing in an array is an effective solution to this problem.
The medium in which biological particles exist is complex.The types and quantity proportions of nontarget particles are usually very large.Using a single separation method to process biological particles in complex media often fails to simultaneously achieve the three important indicators of high purity, high recovery rate, and high particle throughput.A common current strategy is preprocessing the sample, such as dilution and crude (pre) fractionation.These additional preprocessing steps will increase the complexity and time consumption of the separation.However, these pretreatment steps can be fully integrated into microfluidic devices.Thus, using multiple methods for step separation is an effective way to cope with stringent practical application requirements.
In conclusion, taking advantage of the integrability of microfluidic chips is the key to future separation devices.This includes parallel connections of a single technique and series connections of multiple techniques.Additionally, using multiple chips for distributed integration is much more feasible than fully integrating multiple techniques on the same chip.These strategies can improve accessibility by facilitating the commercialization of separated chips/systems so that more researchers can apply them in different fields.

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Microfluidic-based optical separation techniques for biological particles.(A) Separation of particles based on the light scattering force.Reproduced with permission. 123Copyright 2008, American Chemical Society.(B) Separation of particles based on a silicon waveguide-pair array.Reproduced with permission. 124Copyright 2021, Elsevier.(C) Particles are continuously separated using optical tweezers based on fluorescent signals.Reproduced with permission.

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Microfiltration techniques for biological particles.(A) Capture of circulating tumor cell clusters based on meshed microwells.Reproduced with permission. 76Copyright 2022, Springer Nature.(B) Capture of circulating tumor cell clusters based on micropillars.(C) Continuous separation of blood cells from whole blood based on microfiltration membrane.(D) Continuous separation of tumor cells from whole blood based on bioinspired lobe filtration system.

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Dean flow separation techniques for biological particles.(A) Rectangular channel Dean flow device.Reproduced with permission.132Copyright 2013, Springer Nature.(B) Trapezoidal channel Dean flow device.Reproduced with permission.133Copyright 2012, American Chemical Society.(C) Double spiral channel Dean flow device.Reproduced with permission.134Copyright 2013, AIP Publishing.(D) Sheath fluid enhanced Dean flow device.Reproduced with permission.135Copyright 2015, American Chemical Society.WANG ET AL.

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Pinched flow fractionation techniques for biological particles.(A) Traditional pinched flow fractionation device.

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Contraction-expansion array separation techniques for biological particles.(A) Separation based on asymmetric serpentine-curved microchannels.Reproduced with permission.82Copyright 2008, American Chemical Society.(B) Separation based on rectangular contraction-expansion array microchannels.Reproduced with permission.142Copyright 2011, Elsevier.(C) Separation based on triangular contraction-expansion array microchannels.Reproduced with permission.83Copyright 2021, The Royal Society of Chemistry.(D) Separation based on symmetrical serpentine microchannels.Reproduced with permission.143Copyright 2014, Springer Nature.
Summary of biological particle separation techniques based on microfluidics.
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