**7.1. Fundamentals of OPT**

value. The temperature rise of the suspending medium must be kept low to avoid bioparticle

Thermophoretic manipulation of yeast cells has been demonstrated by Lin et al. [46] using low-power and flexible all-optical manipulation method, which presented in **Figure 4b**. They generate light-controlled temperature gradient field thus to trap the suspended cells due to permittivity gradient in the electric double layer of the cell membrane-charged surface. In fact, they manage to realize arbitrary spatial arrangement, as well as precise rotation of single-cell assemblies, with resolution down to 100 nm. J. Chen et al. [48] demonstrate thermophoretic manipulation of *E. coli* by inducing thermal gradient by microscale electric thermal heater. The electric thermal heater is fabricated by gold thin film by means of direct writing with

sample as well as to ensure that the trapping is entirely due to thermal effect. Osterman et al. [49] use microscale thermophoresis to study all potential intraviral protein-protein interactions of hepatitis E virus in order to understand the viral replication cycle. The thermal gradient is generated using 100 V-powered red LED for 25 s radiation. Remarkable thermophoretic manipulation of nucleic acids has been demonstrated by He et al. [50], in which translocation of DNA through nanopore utilizing cross-pore thermal gradient has been achieved. Heating of *cis* chamber is performed by exterior heater to maintain the environment at the melting temperature of the double-strand DNA to transform into single-strand DNA. Extensive temperature drop across the pore is caused by thermal-insulating membrane that separate the *cis* and *trans* chambers, resulting in the thermophoretic translocation of the DNA from *cis* to *trans* chamber. Wolff et al. [51] perform microscale thermophoresis upon different forms of the protein α-synuclein, which is associated with Parkinson's disease, to quantitatively characterize

**Figure 4.** (a) Thermophoresis. Thermophilic bioparticles A (purple) diffuse to regions with higher temperature, while thermophobic bioparticles B (yellow) diffuse to lower temperature regions (Reprinted with permission from Md Ali et al. [13]. Copyright 2016 Royal Society of Chemistry). (b) Manipulation of model organisms by thermophoresis. Optothermal thermophoretic tweezer manipulation is performed by the following: (1) Laser is directed to the digital micromirror device (DMD). (2) Resultant image is focused on gold nanoparticle (AuNP) substrate for surface plasmon excitation. (3) Plasmon-enhanced optothermal potentials defined by the DMD-controlled optical images are exploited to trap and arbitrarily manipulate colloidal particles or biological cells (Adapted with permission from Lin et al. [47].

is coated over the gold thin film to electrically isolate the

damage, such as in the case of DNA, which is from 23 to 31°C [47].

**6.2. Thermophoretic manipulation of bioparticles**

a femtosecond laser. A thin SiO<sup>2</sup>

94 Microfluidics and Nanofluidics

Copyright 2017 American Chemical Society).

Emission of light by a light source induces scattering and gradient forces, which affect particle in the light propagation axis. Scattering force, *Fscat*, affects in the direction of propagation, pushing the particle away from the light source. Gradient force, *Fgrad*, on the other hand, affects the direction of the optical field gradient, attracting the particle to the region with peak spatial light intensity [11] as shown in **Figure 5a**.

Optical tweezing or trapping depends on the dimension range of the particle under manipulation, which is governed by two physical principles, i.e., (1) *Mie* scattering and (2) *Rayleigh* scattering [12]. *Mie* regime condition governs for the condition where the particle dimension range is greater than the wavelength of light (*d >> λ*) which can be explained by ray optics. Cell-type bioparticle (micron-sized) manipulation lies on this regime. Rays of light carry momentum and

**Figure 5.** (a) Optical tweezing or trapping. (Left) Scattering force, *Fscat*, pushes bioparticle away from the light source following the direction of light propagation. Gradient force, *Fgrad*, attracts bioparticle to the spatial light intensity peak according to the direction of the optical field gradient. (Right) Bioparticle is trapped at the highest intensity region, i.e., light focus, as emission of light through a high numerical aperture number lens generates *Fgrad* > *Fscat* condition (Reprinted with permission from Md Ali et al. [13]. Copyright 2016 Royal Society of Chemistry). (b) Manipulation of protein by optical tweezing or trapping: (1) Double-nanohole optical tweezer setup. (2) Details of manipulation of microfluidic device construct. Proteins are trapped at the focus of optical trap at the vicinity of the gold nanohole surface while at the same time unfolding between the double nanoholes. (3) SEM image of the double nanohole (ODF, optical density filter; HWP, half-wave plate; BE, beam expander; MR, mirror; MO, microscope objective; OI MO, oil immersion microscope objective; APD, avalanche photodiode) (Reprinted with permission from Pang and Gordon [52]. Copyright 2011 American Chemical Society).

is refracted when passing through a particle with a refractive index, *n*<sup>2</sup> , which is greater than the surrounding medium, *n*<sup>1</sup> . The rate of the momentum change in the detected rays develops an equal and opposite rate of momentum change of the particle, producing a force by Newton's second law due to conservation of momentum. When a particle is placed in a light gradient, the sum of all rays passing through it creates an imbalance in force, pushing the particle toward the region with higher intensity of light. A focus constructs a trap as the strong light gradient points to the center. *Rayleigh* regime governs for bioparticles with dimension far less than the wavelength (*d << λ*), such as protein and nucleic acids. In this regime, the particles are treated as extremely small point dipoles polarized by a uniform electric field, which then interacts with light field. The dipole moment, *pdipole*, induced by a uniform electric field *E*, is given by

$$p\_{dipole} = 4\pi n\_m^2 \varepsilon\_0 a^3 \left(\frac{m^2 - 1}{m^2 + 2}\right) \text{E} \tag{13}$$

status of cancerous cells. They perform the measurement for human neuroblastoma SK-N-SH and rat C6 glioma cells. Stem cell manipulation has been performed by Kirkham et al. [56]. They develop remarkable holographic optical tweezer and demonstrate the micromanipulation of several stem cells, including mouse embryonic stem cells, mouse mesenchymal stem cells, and mouse primary calvarea cells, as well as microstructures, such as poly(DL-lactic-co-glycolic acid) microparticles and electrospun fiber fragments. They succeeded in accurately construct threedimensional architecture with varying geometries from cocultured cells and microstructure and then stabilized them using hydrogels and cell-cell adhesion methods. Zakrisson et al. [57] perform the optical trapping of nonpiliated strain of *E. coli* bacteria, as well as transformed strain HB101/pHMG93 (expressed as P pili) and the HB101/pAZZ50 strain (expressed as SII pili) in the development of method to determine the presence of pili on a single bacterium. The method comprises of measurement of the bacterium by imaging, then estimation of the fluid drag using an analytical model based on the size, and measurement of the effective fluid drag by oscillating the sample, while the single bacterium is trapped by an optical tweezer. Variation between estimation and measured fluid drag determines the existence of pili. Progress in virus manipulation has been shown by Pang et al. [58]. They conduct optical trapping of individual human immunodeficiency virus (HIV-1) in culture fluid under native conditions and then subsequently perform multiparameter analysis of individual virions, including diameter measurement, concentrationdependent aggregation, and monitoring of viral protein using two-photon fluorescence. Nucleic acid optical manipulation has been achieved by Ngo et al. [59] who use single-molecule assay which integrates fluorescence and optical tweezers generated by infrared laser to manipulate a single nucleosome under force and simultaneously analyze its local conformational transitions. The nucleosome is affixed to a polyethylene glycol (PEG)-coated glass surface at one end of the DNA and pulled via a λ-DNA tethered to the other end by an optical trap to apply force. Pang and Gordon [52], as shown in **Figure 5b**, perform optical trapping and stable unfolding of an individual BSA molecule using a double nanohole in a gold film. The double nanohole with 15 nm separated sharp tips is milled on Au/Ti/glass substrate by a focused ion beam, while the optical trap is generated by 820 nm laser focused onto the sample using a 100× oil immersion microscope objective in a polarization with electric field aligned with the tips of the double

Biological Particle Control and Separation using Active Forces in Microfluidic Environments

http://dx.doi.org/10.5772/intechopen.75714

97

Manipulation of bioparticles in micro-/nanofluidic, integrated with active manipulation mechanisms, i.e., dielectrophoresis, magnetophoresis, acoustophoresis, thermophoresis, and optical tweezing/trapping, has been discussed in this chapter. Description of the underlying fundamental theory is provided at the beginning, and state-of-the-art implementations into a wide range of bioparticles are carefully introduced. DEP has shown rapid progress into exploration of extremely small bioparticle manipulation, i.e., virus, nucleic acids, and protein. In particular, demonstration of DNA sorting [23] and impedance-based protein capturing [25] prove the potential for nanoscale application, as well as genetic and molecular biology studies. MAG is advantageous in selective manipulation benefited by biofunctionalization of magnetic micro-/ nanoparticle for affinity binding to target bioparticles. Novel achievement in customization of

nanohole.

**8. Conclusion and future perspectives**

where *n*1*<sup>m</sup>* and *n*2*<sup>p</sup>* are refractive indices of the suspending medium and the particle, respectively, *a* is the particle radius, *ε*<sup>0</sup> is the vacuum permittivity, and *m* is the contrast ratio of the indices, i.e., *m = np /nm*. The magnitudes of scattering force, *Fscat*, and gradient force, *Fgrad*, based on point dipole interaction with light field method are given by

$$F\_{sant} = \frac{8\pi n\_m}{3c} \frac{k^4 a^4}{\left(m^2 + 2\right)} \left(\frac{m^2 - 1}{m^2 + 2}\right) I \tag{14}$$

$$F\_{gmd} = \frac{2\pi n\_m a^3}{c} \left(\frac{m^2 - 1}{m^2 + 2}\right) \nabla I \tag{15}$$

where *Fscat* is the scattering force, *Fgrad* is the gradient force, *I* is the light intensity, *c* is the speed of light, *k* is the wave number (*k* = 2*π*/*λ*), and *λ* is the wavelength. Trapping is achieved at the highest intensity axis when *Fgrad* > *Fscat*.

OPT can be created by light emission through a high numerical aperture number (NA) of microscope objective (MO), which focuses light tightly and results in a force along the highest intensity axis, but in the backward direction, which causes the bioparticle to be demobilized, as presented in **Figure 5a**.

#### **7.2. Optical tweezing or trapping of bioparticles**

Favre-Bulle et al. [53] use optical trapping in vivo to manipulate otoliths in larval zebrafish to stimulate the vestibular system. Lateral and medial forces upon the otolith cause complementary corrective tail motion, while lateral force on either otolith causes a rolling correction in both eyes. Fascinating manipulation of blood has been demonstrated by Zhong et al. [54], who perform manipulation of RBCs in vivo, i.e., within subdermal capillaries in living mice, using infrared optical tweezers. They demonstrate the optical trapping and three-dimensional manipulation of single RBC in the capillary, as well as multiple RBC trapping, forming capillary blockage and clearance, by turning on and off the optical tweezer. Pradhan et al. [55] use optical trapping to bring a single cell of neural tumor cell lines into close proximity of another and measure the time required for cell-cell adhesion to form, as this method can be used to assess the differentiation status of cancerous cells. They perform the measurement for human neuroblastoma SK-N-SH and rat C6 glioma cells. Stem cell manipulation has been performed by Kirkham et al. [56]. They develop remarkable holographic optical tweezer and demonstrate the micromanipulation of several stem cells, including mouse embryonic stem cells, mouse mesenchymal stem cells, and mouse primary calvarea cells, as well as microstructures, such as poly(DL-lactic-co-glycolic acid) microparticles and electrospun fiber fragments. They succeeded in accurately construct threedimensional architecture with varying geometries from cocultured cells and microstructure and then stabilized them using hydrogels and cell-cell adhesion methods. Zakrisson et al. [57] perform the optical trapping of nonpiliated strain of *E. coli* bacteria, as well as transformed strain HB101/pHMG93 (expressed as P pili) and the HB101/pAZZ50 strain (expressed as SII pili) in the development of method to determine the presence of pili on a single bacterium. The method comprises of measurement of the bacterium by imaging, then estimation of the fluid drag using an analytical model based on the size, and measurement of the effective fluid drag by oscillating the sample, while the single bacterium is trapped by an optical tweezer. Variation between estimation and measured fluid drag determines the existence of pili. Progress in virus manipulation has been shown by Pang et al. [58]. They conduct optical trapping of individual human immunodeficiency virus (HIV-1) in culture fluid under native conditions and then subsequently perform multiparameter analysis of individual virions, including diameter measurement, concentrationdependent aggregation, and monitoring of viral protein using two-photon fluorescence. Nucleic acid optical manipulation has been achieved by Ngo et al. [59] who use single-molecule assay which integrates fluorescence and optical tweezers generated by infrared laser to manipulate a single nucleosome under force and simultaneously analyze its local conformational transitions. The nucleosome is affixed to a polyethylene glycol (PEG)-coated glass surface at one end of the DNA and pulled via a λ-DNA tethered to the other end by an optical trap to apply force. Pang and Gordon [52], as shown in **Figure 5b**, perform optical trapping and stable unfolding of an individual BSA molecule using a double nanohole in a gold film. The double nanohole with 15 nm separated sharp tips is milled on Au/Ti/glass substrate by a focused ion beam, while the optical trap is generated by 820 nm laser focused onto the sample using a 100× oil immersion microscope objective in a polarization with electric field aligned with the tips of the double nanohole.
