**2. Patterning, separation and detection in microfluidics**

In Microfluidics system technology there is an separation methods which can manipulate individual cells having the potential and empowers the experiment sets larger with lower reagent costs and allow for faster reaction work compared to conventional methods [6] for separation and modeling area using micropatterning for the samples like blood and other species like mammalian cells (*K. pneumoniae*) focused on the size and mass of the bioparticle (Spherical and Non-spherical). Nonetheless in recent years Klebsiella species has now become one of the important antibody in infections like nosocomial even there are some important members of Klebsiella breed of Entero-bacteriaceae and some have been exhibited in human laboratory specimens like *K. rhinoscleromatis* and *K. oxytoca* [7]. However separation and detection for the manipulations from the fluidic sample of bioparticles using microfluidics are sensible and challenging issues in laboratories depending on the flow rates, throughput and clogging on microscale [5].

Separation of size-based particle and aborting is occupied in many filtration systems of commonly used tap water filters to a system with complex size separation chromatography systems. As separation of size-based particle has various intent which consists of distillation of fluids or air and analytes concentration (macromolecules or proteins, DNA and others) in separation of components and biological cells. Depending on the size and mass based separation the modeling like patterning or sieving methods are commonly used where it will allow those bioparticles (spherical and non-spherical) to flow through gaps according to hydrodynamic flow rate and based on fluid velocity and viscosity measurement the other particles can be sorted individually for precise outcome [8].

#### **2.1. Bioparticles of spherical and non-spherical shape**

Ideally for geometry, it has a definite shape associated with the index of shape for adequate characterization. Depending on the characteristics of the bioparticles and its recognition visually is straightforward as defining in words or numerical value of a shape can be in a limited portion. Despite the efforts made in previous works in evolving a sphericity or index have created many hurdles for distributing values on the 3-dimensional complex irregular shape [9–11]. For the shape factor there is no perturbation with it for changes in the drag force of particle for spherical and non-spherical as there has been a development in the dynamic shape factor relatively. This ratio of drag force is calculated for the forces experienced by the spherical or nonspherical particle over the ratio of other particles with the volume equivalently flowing with the medium and same flow velocity. Other than spherical particle there is a an phenomenon change in the non-spherical particle as understanding the indexing of non-spherical particle is important with the chemical, physical, biological functions and other phenomenon by which the changes in the effect of bioparticles cannot be denied. However the anatomy of some proteins molecule to the utmost dimensions of galaxies and stars neglecting the effect of shape can be made. Interaction with the analysis of particles deeply requires some considerations of shape for behavior in the system of the particle in applications practically to conclude [8] (**Figure 1**).

#### **2.2. Techniques for sorting**

BioMEMS has different types of devices which determines that device for this term have some manipulations in chemicals for about smallest part in form of microlitre for bacteria or proteins separation purpose of different cells (spherical and non-spherical) drug delivery and detection of contaminant with other manipulations necessary. However, some of the micro electro mechanical system (device instrument), which is attached to normal surgical instruments, is also called BioMEMS type but it is not included in such normal devices due to restriction honored and considered as technical type instrument. There are some other devices which involve itself under BioMEMS to accentuate the idea and perform all tasked from input of samples to the detection of the cells or proteins named *micro-total-analysis system.* Operations at chip scale can replace some familiar works of laboratory process known as *lab-on-a-chip* as well as for the approach for conducting measurements parallel an *array processor* are been included. There are some other term which does not belongs to the BioMEMS but

A splendid addition has been exhibited which allows for the sorting depending on selection and different interests in analytes [2–4]. Thus this chapter deals with the different techniques used for separation and for micropatterning of array channels used in different applications of biomedicals and other BioMEMS terms. First part of this chapter deals with the active and passive approaches for bioparticles and cellular separation depending on the fluid velocities and its concentration depending on applications and designing. The second half of the chap-

In Microfluidics system technology there is an separation methods which can manipulate individual cells having the potential and empowers the experiment sets larger with lower reagent costs and allow for faster reaction work compared to conventional methods [6] for separation and modeling area using micropatterning for the samples like blood and other species like mammalian cells (*K. pneumoniae*) focused on the size and mass of the bioparticle (Spherical and Non-spherical). Nonetheless in recent years Klebsiella species has now become one of the important antibody in infections like nosocomial even there are some important members of Klebsiella breed of Entero-bacteriaceae and some have been exhibited in human laboratory specimens like *K. rhinoscleromatis* and *K. oxytoca* [7]. However separation and detection for the manipulations from the fluidic sample of bioparticles using microfluidics are sensible and challenging issues in laboratories depending on the flow rates, throughput

Separation of size-based particle and aborting is occupied in many filtration systems of commonly used tap water filters to a system with complex size separation chromatography systems. As separation of size-based particle has various intent which consists of distillation of fluids or air and analytes concentration (macromolecules or proteins, DNA and others) in separation of components and biological cells. Depending on the size and mass based separation the modeling like patterning or sieving methods are commonly used where it will allow

ter focuses on the simulations of the sorting and the detection techniques [5].

**2. Patterning, separation and detection in microfluidics**

and clogging on microscale [5].

it emerges with it are defined with self as *microfluidics* [1].

72 MEMS Sensors - Design and Application

In recent years for studies of application on cell separation there were many techniques extended which emerged in the field of microfluidics for biomolecular analysis which are initially driven as per requirement. While manipulation and Cell separation is a crucial sample step for processing in many medical assays and low Reynolds numbers as well as predictable flows, small fluid volumes, small dimensions, materials and the established microfabrication

**Figure 1.** Types of bacteria present with its size of shape [12].

techniques which are typical microfluidic device factors allows user to work with cells on microscale. Existing microfluidic separation methods are categorized into two methods as active and passive methods where active methods incorporate an external force and passive methods rely on carefully designed channel geometries and internal forces to sort different particles. Classification is as shown in **Figure 2**.

Hydrodynamic force is one of the inherent physical principle and basic in system of microfluidics where inertial force and dean rotation force has been utilized, though hydrodynamic force based separation system can be created by fluid dynamic theory based microchannel network. An Acoustic method utilizes the ultrasonic standing waves and allowing a manipulation of cells then other separation methods like dielectrophoretic, magnetic and others. The basic principle of acoustic sorting is to use pressure gradients generated by ultrasonic standing waves since most microfluidic system uses liquid medium as the working fluid. DEP is the electro kinetic motion which occurs when polarisable particle is placed in non-uniform electric fields and particle motion induced by DEP force is influenced by the ambient electric field and the properties of electric particles or solutions. Magnetic sorting system can be of two different categories for separation (I) Attaching magnetic particles to the cells to react with the magnetic field and (II) Utilizing the native magnetic properties of the cells in laminar flow for some basic motion of particles expressed as Newton's second law [17] schematics

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Microfiltration is a method of basic concept for separating and sorting microparticles which utilizes the size of micropores and the gap between microposts as a lattice or sieve. Accordingly, microfiltration is highly dependent on the sizes of the microparticles. The advantages of this methods discussed are easy for understanding the separation principles for engineer or scientists to implement for an application to existing methods [17]. Thus by this study we have introduced a developed technique HDF which focuses on the shear flow of input associated with buffer streams which constricts the stream flow within the middle of the microchannel

**Figure 3.** Cell Separation principle schematics (a) Geometrical constraints for cell size in microfiltration technique (b) hydrodynamic forces applied to the cells in the network of microchannel (c) Between the magnetic force field and the magnetic particles attached to the cells they are separated by the attraction force (d) The ultrasonic waves generated by the transducers from which acoustic cell separation utilizes acoustic primary radiation force (e) The electro kinetic

motion of polarisable cells in non-uniform electric fields is utilized by Dielectrophoretic force (DEP) [17].

shown in **Figure 3**.

In this chapter, we introduce various principles and related methods including some common separation methods for active type include immunomagnetic separation (IMS), acoustophoresis, electrophoresis, dielectrophoresis, optical force and flow cytometry or FACS [14]. There are different technologies or methods in microfluidics for precise, passive and continuous sorting systems invented including Hydrodynamic inertial force, Deterministic Lateral Displacement (DLD), Pinched-Flow Fractionation (PFF) and Hydrodynamic Filtration (HDF) [15, 16].

Microfluidic technologies that can manipulate individual cells with the potential enable larger experiment sets, lower reagent costs and allow for faster reaction work compared to conventional methods [6]. PFF is a separation technique where a field is applied to a fluid suspension pumped through channel which is narrow and long, flow in perpendicular direction depending on their differing "Flexibility" under the exerted force by the field and to cause separation of the particles present in the fluid. In recent years a number of microfluidic devices have been advanced for the continuous separation of bioparticles by employing unique techniques. One of the prominent separation method is DLD in which particles drives through the post to post arrays positions. The interaction of the different particles with different size and post to post array directions leads to different bacteria to drift in different directions with respect to the arrays thus causing the continuous fractionation and two dimensional of the sample mixture.

**Figure 2.** Classification of microfluidic sorting and separation techniques [13].

Hydrodynamic force is one of the inherent physical principle and basic in system of microfluidics where inertial force and dean rotation force has been utilized, though hydrodynamic force based separation system can be created by fluid dynamic theory based microchannel network. An Acoustic method utilizes the ultrasonic standing waves and allowing a manipulation of cells then other separation methods like dielectrophoretic, magnetic and others. The basic principle of acoustic sorting is to use pressure gradients generated by ultrasonic standing waves since most microfluidic system uses liquid medium as the working fluid. DEP is the electro kinetic motion which occurs when polarisable particle is placed in non-uniform electric fields and particle motion induced by DEP force is influenced by the ambient electric field and the properties of electric particles or solutions. Magnetic sorting system can be of two different categories for separation (I) Attaching magnetic particles to the cells to react with the magnetic field and (II) Utilizing the native magnetic properties of the cells in laminar flow for some basic motion of particles expressed as Newton's second law [17] schematics shown in **Figure 3**.

techniques which are typical microfluidic device factors allows user to work with cells on microscale. Existing microfluidic separation methods are categorized into two methods as active and passive methods where active methods incorporate an external force and passive methods rely on carefully designed channel geometries and internal forces to sort different

In this chapter, we introduce various principles and related methods including some common separation methods for active type include immunomagnetic separation (IMS), acoustophoresis, electrophoresis, dielectrophoresis, optical force and flow cytometry or FACS [14]. There are different technologies or methods in microfluidics for precise, passive and continuous sorting systems invented including Hydrodynamic inertial force, Deterministic Lateral Displacement (DLD), Pinched-Flow Fractionation (PFF) and Hydrodynamic Filtration (HDF) [15, 16].

Microfluidic technologies that can manipulate individual cells with the potential enable larger experiment sets, lower reagent costs and allow for faster reaction work compared to conventional methods [6]. PFF is a separation technique where a field is applied to a fluid suspension pumped through channel which is narrow and long, flow in perpendicular direction depending on their differing "Flexibility" under the exerted force by the field and to cause separation of the particles present in the fluid. In recent years a number of microfluidic devices have been advanced for the continuous separation of bioparticles by employing unique techniques. One of the prominent separation method is DLD in which particles drives through the post to post arrays positions. The interaction of the different particles with different size and post to post array directions leads to different bacteria to drift in different directions with respect to the arrays thus causing the continuous fractionation and two dimensional of the sample mixture.

particles. Classification is as shown in **Figure 2**.

74 MEMS Sensors - Design and Application

**Figure 2.** Classification of microfluidic sorting and separation techniques [13].

Microfiltration is a method of basic concept for separating and sorting microparticles which utilizes the size of micropores and the gap between microposts as a lattice or sieve. Accordingly, microfiltration is highly dependent on the sizes of the microparticles. The advantages of this methods discussed are easy for understanding the separation principles for engineer or scientists to implement for an application to existing methods [17]. Thus by this study we have introduced a developed technique HDF which focuses on the shear flow of input associated with buffer streams which constricts the stream flow within the middle of the microchannel

**Figure 3.** Cell Separation principle schematics (a) Geometrical constraints for cell size in microfiltration technique (b) hydrodynamic forces applied to the cells in the network of microchannel (c) Between the magnetic force field and the magnetic particles attached to the cells they are separated by the attraction force (d) The ultrasonic waves generated by the transducers from which acoustic cell separation utilizes acoustic primary radiation force (e) The electro kinetic motion of polarisable cells in non-uniform electric fields is utilized by Dielectrophoretic force (DEP) [17].

accommodated with particles in the plane of sidewalls with narrow width [18] where both concentration and classification of particles can be examined at the same interval of time by introducing a solution consisting of particle beads of various mass and size.

We preceded here with a method that has been adopted to invent a separation mechanism, Continuous processing, high precision for particle separation and high throughput [17]. When a particle flows in a microchannel the center position of the particle cannot remain present on a certain distance from sidewalls which is equal to the particle radius. The method of filtration utilizes this fact and is performed using a Sieve type-shaped microchannel network having multiple side branch channels/sieve-shaped networks. According to flow rates in microchannel by fluid flow (μl) it withdraws a small amount of liquid continuously on intervals from the main stream through the side microchannel network and particles are concentrated and aligned in the network according to size and mass. However the concentrated and sorted particles can be collected according to size and mass through all other output channels in the stream of the microchannel network. Therefore continuous introduction of a particle suspension into the microchannel enable particle sorting, concentration and classification at the same time with precision [19]. Whenever there is a difference in particle size and mass then separation becomes difficult for the result and mesh clogging is inevitable [20].

Hydrodynamic Filtration is one of the most frequently used technique to classify particles suspended in fluid flow due to sedimentation. Existing filtration methods performed either in batch or continuous manner and large-scale treatment can be easily achieved. Biological entities such as rod-shaped bacteria and disc-shaped red blood cells (RBCs), disproportional length and width which complicate the separation process designed for spherical particles and the narrowest width has to be considered for the separation criteria within the design parameters of the microfluidic devices. Some of the examples for cell separation include (**Figures 4** and **5**):


However there are some essential approaches through which separation takes in microfluids, • From a biological sample if a single type cell is removed it can be called as **Depletion region.**

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**Figure 5.** Sorting and separation for different cells from mixed population to isolated populations [20].

• Similarly when there is need of removing cells other then the single cell like RBC a **Negative selection** can be done where it will leave single cell type and other packet of cell needed to be removed can be separated. Example like bone marrow or whole blood sample, removal

• Whenever there is any downstream analysis then a mechanism of removal cell type is targeted whichever cell depending on size and mass can be separated this type of typical selection can be processed by **positive selection**. This type can be possible in monoclonal antibodies and performed by aiming a surface marker of cells (biopar-

Example mononuclear cells from which RBCs can be removed.

of cells varying with size and others.

ticles) [20].

• Resistant cells isolation from peripheral blood

**Figure 4.** Role of microfluidics for the separation.

**Figure 5.** Sorting and separation for different cells from mixed population to isolated populations [20].

However there are some essential approaches through which separation takes in microfluids,


**Figure 4.** Role of microfluidics for the separation.

• Blood cells (WBC) isolation from tissue

76 MEMS Sensors - Design and Application

• Circulating Tumor Cells (CTC) from blood

• Resistant cells isolation from peripheral blood

accommodated with particles in the plane of sidewalls with narrow width [18] where both concentration and classification of particles can be examined at the same interval of time by

We preceded here with a method that has been adopted to invent a separation mechanism, Continuous processing, high precision for particle separation and high throughput [17]. When a particle flows in a microchannel the center position of the particle cannot remain present on a certain distance from sidewalls which is equal to the particle radius. The method of filtration utilizes this fact and is performed using a Sieve type-shaped microchannel network having multiple side branch channels/sieve-shaped networks. According to flow rates in microchannel by fluid flow (μl) it withdraws a small amount of liquid continuously on intervals from the main stream through the side microchannel network and particles are concentrated and aligned in the network according to size and mass. However the concentrated and sorted particles can be collected according to size and mass through all other output channels in the stream of the microchannel network. Therefore continuous introduction of a particle suspension into the microchannel enable particle sorting, concentration and classification at the same time with precision [19]. Whenever there is a difference in particle size and mass then separa-

Hydrodynamic Filtration is one of the most frequently used technique to classify particles suspended in fluid flow due to sedimentation. Existing filtration methods performed either in batch or continuous manner and large-scale treatment can be easily achieved. Biological entities such as rod-shaped bacteria and disc-shaped red blood cells (RBCs), disproportional length and width which complicate the separation process designed for spherical particles and the narrowest width has to be considered for the separation criteria within the design parameters of the microfluidic devices. Some of the examples for cell separation include (**Figures 4** and **5**):

• Separation of some bacteria from food which are pathogenic to health and other systems

introducing a solution consisting of particle beads of various mass and size.

tion becomes difficult for the result and mesh clogging is inevitable [20].
