**5. Applications of magnetic fields in lab-on-a-chip**

Once provided the reader with an overview of physics at the microscale, as well as of magnetic materials properties and principles to be exploited, some real applications of LOCs will be described, where using magnetic fields. Such applications range from biological samples handling to chemical reactions and other manipulation tasks. As a consequence, in order to better analyze the potential of using magnetic fields in this context, it is useful to classify the applications in three main areas: (1) on-chip bioanalysis; (2) cell separation or manipulation; and (3) non-conventional manipulation techniques.

#### **5.1. On-chip bioanalysis**

2 0

m

*r N*

*M*

46 Lab-on-a-Chip Fabrication and Application

coils, respectively.

(13) and (14) as follows:

2

*M MM*

= -

3 3 2 2 2 2

*<sup>r</sup>* <sup>=</sup> *<sup>I</sup> <sup>B</sup>* (15)

*<sup>r</sup>* <sup>=</sup> *<sup>I</sup> <sup>B</sup>* (16)

*<sup>I</sup> <sup>B</sup>* (14)

2 2

NH,IH,rH, NM,IM,rM are the numbers of windings, current, and radius of Helmholtz and Maxwell

When considering a combination of Helmholtz and Maxwell coils (**Figure 4A**), the magnetic field B and magnetic field gradient ∇B in the workspace can be derived analytically by Eqs.

> <sup>0</sup> 8 5 5 *H H H*

48 3 49 7

obtain both a uniform field gradient and magnetic field uniformity [29].

*µ N*

0 2

*µ N*

Equations (15) and (16) clearly show that Helmholtz coils are able to generate a uniform magnetic field, whereas Maxwell coils produce a uniform magnetic field gradient along its axis. For this reason, combinations of Helmholtz and Maxwell coils have been exploited to

Nonuniform field setups have been developed as well. Despite the major complexity both in terms of design/fabrication and control, they enable an increase in the number of controlled degrees of freedom. In this sense, a representative example is the OctoMag system [30, 31] (**Figure 4B**), designed for the control of intraocular microrobots for minimally invasive retinal therapy and diagnosis, but showing also potentialities for use as a wireless micromanipulation apparatus. It consists of eight stationary electromagnets with soft-magnetic cores able to generate predefined values of magnetic field and gradient, providing the manipulated object with five degrees of freedom; this system can operate closed-loop position control by exploiting computer-assisted visual tracking or in open loop by relying only on the operator microscopemediated visual feedback. Alternative approaches aim at exploiting other sources of nonuni‐ form magnetic fields: Martel et al. [32] demonstrated the effectiveness of using an MRI scanner for the control of a swarm of magnetotactic bacteria in executing a manipulation task on microobjects. Micro-assembly of micro-objects using a cluster of microparticles (with average diameter of 100 μm) and a magnetic-based manipulation system has also been shown in [33].

*M M M*

1 1

æ ö ç ÷

2 2

é ùé ù æö æö ê úê ú +- ++ ç÷ ç÷ èø èø è ø ë ûë û

*d d r zr z*

A vast number of reactions in genomics, proteomics, and clinical medicine need molecular mixing of fluids or recognition events between single strands of DNA, between antibodies and antigens, or between receptors and cells. Such reactions usually require a number of steps that must be performed sequentially, such as isolation, washing, or purification. In this kind of applications, the introduction of automation could lead to higher throughput and the use of magnetic fields, commonly mediated by the use of magnetic particles, has revealed to be extremely useful in some on-chip functions such as the mixing of fluids, selective capture of analytes, later to be transferred for further analysis steps, or the performance of stringency and washing. Usually two main properties of magnetic particles are exploited in analytical assays: the possibility to biofunctionalize them, thus enabling selective binding and related applica‐ tions, and the capability to form supra-particle structures, such as chains, exploited mainly in fluid mixing and analytes capture applications [5].

Usually, magnetic particles are labeled with molecules, for example, antibodies, showing high affinity for the target species and able to mediate the binding with them. Often multistep binding processes are carried out to bind the particle–analyte complex, for example to fluorescent dies, thus to enable target detection [34]. In other cases, for additional purification steps after labeling, magnetic separation, mediated by magnetophoresis phenomena, or transfer processes are required to enable further purification/washing steps or analysis. This kind of procedures can be exploited both for the in vitro purification of nucleic acids or proteins and for biomolecules separation, as well as DNA sequencing. For example, in case of DNA extraction and separation processes, magnetic particles are firstly held in place by exploiting external magnetic fields, thus exposing their functional groups to specific DNA strands. Magnetic separation steps can consequently be performed to isolate the strands of interest from the rest of the sample [35]. In other cases, the target-binding capabilities, with analytes showing at least two epitopes, have been exploited to create large aggregates for albumin detection in buffer [36]: The binding between particles is mediated by the target molecule and thus the extent of the aggregate represents a measure of analytes concentration within the solution and the magnetic properties of the aggregate allow their detection. In this case, non-specific particle clustering should be avoided.

Another interesting application is in the field of biosensing or surface binding bioassays: In this context, magnetic particles can be exploited as binding mediators between the target species and a functionalized surface. The most common configuration in this field is the sandwich one, in which the target molecule binds first the magnetic particles dispersed in the solution. Later, by exploiting magnetic field gradients, the complex can be moved toward the surface of the sensor where interactions at the molecular scale occur [37].

Whereas in detection applications, non-specific magnetic interaction within particles has to be avoided, there are applications in which the capability of magnetic particles to interact each other thanks to magnetic forces to form supra-particle structures can be advantageously exploited. When working with really precious/expensive fluids, really small volumes and microfluidic devices are employed. Due to the small scale and to the strong viscous force, fluid mixing is not straightforward. In this case, magnetic particles can be exploited to steer fluids. This is done specifically for supra-particle structures such as chains created by the dipole– dipole interactions between magnetic particles, and actuated, for example, by means of rotating magnetic fields [38].

#### **5.2. Magnetic cell separation**

Cell manipulation by means of magnetic fields relies on cells magnetic labeling or, alterna‐ tively, on magnetic particles internalization through magnetic field-mediated transfection mechanisms (magnetofection). In a typical in vitro magnetofection system, target cells are located at the bottom of a fluidic chamber well of a culture plate, and a permanent magnet beneath the chamber provides a magnetic force that attracts the biofunctional particles toward the cells.

Separation and isolation of rare cell populations from a heterogeneous suspension is essential for many applications, ranging from disease diagnostics to drug screening. Various separation techniques have been proposed, but magnetic fields emerged as very promising also in this kind of application thanks to the exploitation of the magnetic separation principles presented in the previous section.

Magnetic cell sorting can be operated in either a serial or a parallel manner, resulting in higher throughput with up to 1011 cells processed in 30 min. This process can be operated in both batch and continuous flow mode. In batch processing, the hardware is very simple, including a magnetic field source placed close to a column containing the cells to be separated. Several architectures were developed to this aim both at large scales, exploiting for example ferro‐ magnetic columns [39] and at smaller scales with arrays of electrical wires exploited to produce local magnetic fields [40]. In the case of continuous flow cell sorting, instead, typical magne‐ tophoresis principles are exploited.

kind of procedures can be exploited both for the in vitro purification of nucleic acids or proteins and for biomolecules separation, as well as DNA sequencing. For example, in case of DNA extraction and separation processes, magnetic particles are firstly held in place by exploiting external magnetic fields, thus exposing their functional groups to specific DNA strands. Magnetic separation steps can consequently be performed to isolate the strands of interest from the rest of the sample [35]. In other cases, the target-binding capabilities, with analytes showing at least two epitopes, have been exploited to create large aggregates for albumin detection in buffer [36]: The binding between particles is mediated by the target molecule and thus the extent of the aggregate represents a measure of analytes concentration within the solution and the magnetic properties of the aggregate allow their detection. In this case, non-specific particle

Another interesting application is in the field of biosensing or surface binding bioassays: In this context, magnetic particles can be exploited as binding mediators between the target species and a functionalized surface. The most common configuration in this field is the sandwich one, in which the target molecule binds first the magnetic particles dispersed in the solution. Later, by exploiting magnetic field gradients, the complex can be moved toward the

Whereas in detection applications, non-specific magnetic interaction within particles has to be avoided, there are applications in which the capability of magnetic particles to interact each other thanks to magnetic forces to form supra-particle structures can be advantageously exploited. When working with really precious/expensive fluids, really small volumes and microfluidic devices are employed. Due to the small scale and to the strong viscous force, fluid mixing is not straightforward. In this case, magnetic particles can be exploited to steer fluids. This is done specifically for supra-particle structures such as chains created by the dipole– dipole interactions between magnetic particles, and actuated, for example, by means of

Cell manipulation by means of magnetic fields relies on cells magnetic labeling or, alterna‐ tively, on magnetic particles internalization through magnetic field-mediated transfection mechanisms (magnetofection). In a typical in vitro magnetofection system, target cells are located at the bottom of a fluidic chamber well of a culture plate, and a permanent magnet beneath the chamber provides a magnetic force that attracts the biofunctional particles toward

Separation and isolation of rare cell populations from a heterogeneous suspension is essential for many applications, ranging from disease diagnostics to drug screening. Various separation techniques have been proposed, but magnetic fields emerged as very promising also in this kind of application thanks to the exploitation of the magnetic separation principles presented

Magnetic cell sorting can be operated in either a serial or a parallel manner, resulting in higher throughput with up to 1011 cells processed in 30 min. This process can be operated in both

surface of the sensor where interactions at the molecular scale occur [37].

clustering should be avoided.

48 Lab-on-a-Chip Fabrication and Application

rotating magnetic fields [38].

**5.2. Magnetic cell separation**

in the previous section.

the cells.

Multicell sorting systems rely on the variation in the uptake of magnetic material between different cell populations and thus on the different path deviation produced by magnetic field gradients. They can be also used for the separation of different cell species from heterogeneous samples [41].

The separation of a specific class of cells from a certain sample is extremely important for some applications, for example, for the detection of pathology or for the testing of a therapeutic strategy. For example, diagnosis and treatment of HIV disease rely on the efficient separation of human T-lymphocytes from whole blood [42], whereas in the diagnosis and treatment of malaria, the detection of infected red blood cells (RBCs) and their separation from healthy cells is mandatory [43]. Separation of neuronal cells has gained interest for its potential applications in cell replacement therapy of neurodegenerative disorders such as Parkinson's disease, multiple sclerosis, and Alzheimer's disease [44]. Cell separation methods are also needed for separating nucleated RBCs from the peripheral blood of pregnant women, for monitoring maternal, fetal, and neonatal health [45].

Magnetic field-based cell counting techniques have also been developed. One method estimates the location and number of cells tagged by measuring the magnetic moment of the microsphere tags [46], while another uses a giant magnetoresistive sensor to measure the location of microspheres attached to a surface layered with a bound analyte [47].

#### **5.3. Non-conventional manipulation strategies based on magnetic fields**

When high sensitivity, not compatible with magnetophoretic techniques is required, and independence on the human operator are desirable, microrobotic manipulators acting at the cellular scale can offer significant benefits. Wirelessly controlled (i.e., untethered) cell-sized robots are highly noninvasive. At this length scale, where viscous fluid forces dominate inertial ones, mobile microrobots cause very little mixing or agitation of the surrounding environment. This is a significant advantage, for example, over suction pipetting for life scientists, since pipettes cause relatively large fluid disturbances [48]. Magnetic control of microrobots and microgrippers is gaining growing importance in micro-object manipulation: in addition to increasing the manipulation accuracy, the exploitation of such micro-systems avoids some‐ times the direct magnetization of the sample, through internalization or labeling, thus helping in keeping its integrity. Many challenges have to be faced to enable single cell manipulation. When working with single cells or with really fragile samples, in fact, it is essential to have microstructures with sizes comparable to those ones of the target, to be able to finely control them within the workspace, and to avoid to affect cell viability or samples integrity due to the microrobot exploitation.

Some research groups focusing on microtechnologies have been working toward a high efficiency in vitro fertilization (IVF) process [49] (**Figure 5A**). The IVF goal is to fertilize oocytes, and it consists of several manually or teleoperated manipulation steps that require important practical skills. Sakar et al. [50] developed microtransporters using a simple, single-step microfabrication technique allowing parallel fabrication. They demonstrated that the micro‐ transporters can be navigated to separate individual targeted cells with micron-scale precision and deliver microgels without disturbing the cells in the neighborhood and the local micro‐ environment. Yamanishi et al. [51] presented an innovative driving method, devised for cell sorting, for an on-chip robot actuated by permanent magnets in a chip, where a piezoelectric ceramic is applied to induce ultrasonic vibration to the microfluidic chip and the highfrequency vibration reduces significantly the effective friction on a magnetically driven microtool.

Other interesting magnetic microstructures, devised for cell manipulation in in vitro environ‐ ments for LOC applications, but finally eligible in the future for in vivo applications, have been recently proposed. Examples are novel microgrippers, in which both the navigation and the gripper actuation rely on magnetic fields [52] (**Figure 5B**), 3D laser lithography microcages devised to act as cell carriers (**Figure 5D**) [53] or thin magnetic films working at the air fluid

**Figure 5.** Overview of non-conventional manipulation systems for microrobotics and LOC applications. (A) Conceptu‐ al overview of a microfluidic cell manipulation system based on magnetically driven microtools and exploited for oo‐ cytes handling [49] (reproduced with permission from Royal Society of Chemistry); (B) Schematic representation of a remotely controlled microgripper exploiting magnetic fields both for navigation and for gripper actuation (adapted from [52] and reproduced with permission from Royal Society of Chemistry); (C) SEM image of a magnetic thin film devised for cell manipulation (left) and schematic representation of the film structure with microscope images showing T24 cell compatibility with the magnetic structure (right) [13] (reproduced with permission from Springer); (D) SEM image (above) and confocal microscope image of a magnetic microcage after cell culture [53]; (E) Experimental setup for magnetic micromanipulation (left) and microscope images of the magnetic microrobot during christal manipula‐ tion tasks (adapted from [55] and reproduced with permission of the International Union of Crystallography).

interface and exploiting surface tension phenomena together with magnetic navigation and showing compatibility with cell manipulation applications [54] (**Figure 5C**). Interesting is also the development of manipulation strategies for precise non-contact handling of small and fragile samples based on complex control algorithms aiming at creating vortexes, as demon‐ strated for crystal harvesting applications (**Figure 5E**) [55].
