Principles and applications of medical nanotechnology devices

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Principles and applications of medical nanotechnology devices

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Kamalesh Chaudhari⁎, Swathi Chaudhari⁎, Chandra Prakash Sharma† Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB, Canada* Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal, India†

­C HAPTER OUTLINE 13.1 Introduction..................................................................................................... 276 13.2  Imaging Nanodevices....................................................................................... 276 13.2.1  Applications of Imaging Nanodevices in Drug Delivery.................... 276 13.2.2  Major Imaging Nanodevices and Techniques for Their Observations.. 277 13.3  Nanodevices for the Separation of Biomolecules and Cells................................. 283 13.3.1 Introduction............................................................................... 283 13.3.2  Size- and Charge-Based Separation (Nano-Based).......................... 283 13.3.3  Magnetic Nanoparticles for Separation and Other Applications........ 283 13.3.4  Flow Cytometers......................................................................... 284 13.4 Nano-MEMS..................................................................................................... 287 13.4.1  Introduction to MEMS................................................................. 287 13.4.2  Fabrication of MEMS................................................................... 287 13.4.3  MEMS and Drug Delivery............................................................. 288 13.5  Microfluidic Devices........................................................................................ 289 13.5.1  Introduction to Microfluidic Devices.............................................. 289 13.5.2  Fabrication Techniques for Microfluidic Devices............................. 289 13.5.3  Microfluidic Devices for Drug Delivery........................................... 291 13.6  Interfacing Nanoelectronics With Biology.......................................................... 291 13.6.1  Introduction to Nanoelectronic Devices......................................... 291 13.6.2  Applications of Nanoelectronics in Biology and Drug Delivery.......... 292 13.7 Nanorobots...................................................................................................... 293 13.7.1  Introduction to Nanorobots.......................................................... 293 13.7.2  Nanorobots for Cell Repairing and Drug Delivery............................ 293 13.8 Summary......................................................................................................... 294 References............................................................................................................... 294

Drug Delivery Nanosystems for Biomedical Applications. https://doi.org/10.1016/B978-0-323-50922-0.00013-4 © 2018 Elsevier Inc. All rights reserved.

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13.1 ­INTRODUCTION As it is evident from previous chapters, the area of drug delivery using various nanosystems has developed well in the past few decades. A variety of nanodrug delivery systems (nano-DDS) have been developed for this purpose [1]. Moreover, there are multiple applications of nanotechnology devices which play important roles in the development of drug delivery devices and their assessment. Apart from actual drug delivery nanosystems, nanoparticles can be used for detection, separation, tracking of biomolecules, or drug molecules inside mammalian cells [2]. It is also necessary sometimes to use various nanoprobes to study interaction between drug molecules and targeted biomolecules [3]. Such observations are necessary for the development of fundamental understanding about the behavior of nano-DDS in vivo environment [4]. A major requirement in such investigations is well-engineered nanoscale imaging devices that can be conjugated with the molecule of interest suitable for long-term imaging in vitro as well as in vivo. Nanoscale imaging devices produce plasmonic or fluorescent signals that can be detected using advanced microscopic techniques [3a,5]. Other classes of nanoscale imaging devices include contrast agents for magnetic resonance imaging (MRI) or similar computed tomographic (CT) imaging techniques. Apart from this, various nanodevices have been developed for sensing applications required at the various stages in nano-DDS development. Sensors can be used to detect drug-target binding, understand the effect of cellular environments on drug molecules, or to quantify the interaction of drug on target biomolecules [6]. In many such applications, it is necessary to work with the biological samples of microliter volumes. For this purpose, microfluidic devices have been developed [7]. These devices can mix or separate biomolecules of interest. Sensors can be integrated into microfluidic devices for miniaturizing and minimizing the need of equipment. This is helpful in not only reducing the volume of sample but also in reducing the cost. While these devices and nanosensors are continuously under development, recent advances in research are also focusing on the development of nanorobots for in situ repairing and manipulation of cells as well as onsite delivery of drugs [8]. Nanorobots can be constructed from biological components or nonbiological inorganic micro/ nanostructures [9]. Recent developments in the area of nanorobotics are crucial in defining future of nano-DDS for biomedical applications. Herein this chapter we discuss principles and concepts behind the development of aforementioned nanodevices and provide details about their applications in the area related to drug delivery.

13.2 ­IMAGING NANODEVICES 13.2.1 ­APPLICATIONS OF IMAGING NANODEVICES IN DRUG DELIVERY In the treatment of various diseases or internal body organs it is necessary to develop drugs that target specific locations inside body. Targeting ability of such drug molecules need to be tested and verified. Hence, while developing new nano-DDS, it

13.2 ­ Imaging nanodevices

is always necessary to track and localize the same inside cells and organs. For such tracking and localization experiments in initial stages, nanoscale imaging devices are used for in vitro experiments [10]. Such imaging experiments can provide information about localization of drugs or target molecules in intracellular compartments and organelles. This information can be then used to understand and improve the targeting ability of drugs. In such experiments, fluorescent dye molecules are major labeling agents to track the path of targeting moieties. But fluorescent dye molecules suffer from photobleaching upon long exposure to the excitation light and observations related to tracking cannot be performed over extended period of time [11]. Issue of photobleaching can be solved by using other labeling agents such as fluorescent semiconductor quantum dots or plasmonic nanoparticles. Compositions of quantum dots include sulfides or selenides of lead, cadmium, indium, etc. [12]. Quantum dots can provide stable fluorescence for tracking experiments but due to their toxic composition they can affect the behavior of biological systems [13]. Plasmonic nanoparticles are most stable and efficient labels for long-term imaging experiments [14]. Especially when plasmonic nanoparticles made of noble metals such as gold are used for labeling, they exhibit high signal-to-noise ratio due to their intense plasmonic scattering [15]. Recently developed noble metal quantum clusters (NMQCs) of gold stabilized by protein templates are also good candidates for such labeling applications [16]. Such nanolabels are not only necessary for in vitro experiments but also for the imaging of drug targeting and localization studies in  vivo. Many different types of labels are required in such applications. Nanolabel selection for application is based on compatibility of the same to the testing environment and imaging technique. These techniques can be magnetic resonance-based imaging, NIR fluorescence imaging, etc. For these techniques, special contrast agents are necessary. In subsequent sections below we discuss some of the major nano-bio labels and contrast agents in detail.

13.2.2 ­ MAJOR IMAGING NANODEVICES AND TECHNIQUES FOR THEIR OBSERVATIONS ­Fluorescent nanoparticles

Fluorescence is based on the absorption and emission of electromagnetic radiation by molecules/particles [17]. As shown in Fig. 13.1A, these molecules possess discrete electronic energy levels. Electrons from highest occupied molecular orbital (HOMO) can be excited to lowest unoccupied molecular orbital (LUMO) by irradiating fluorescent moieties with high-energy photons typically of UV (ultra violet) light. Upon excitation, fluorescent molecules in ground state are excited to singlet state, which when returns to ground state, light of lower energy is emitted [18]. Excited molecules/particles can return to ground state through certain transitions, which lead to emission of low energy red, green, or blue photons. Fluorophores for biological imaging experiments are selected on the basis of their following properties: (i) quantum yield, this parameter defines number of photons emitted per number of photons absorbed and signifies the efficiency of fluorophore, (ii) lifetime, this is the average

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UV photon (excitation)

Detector Emission filter

Red photon Green photon

Dichroic mirror Blue photon Excitation filter Objective

(A)

(B)

Specimen

FIG. 13.1 (A) Energy diagram for fluorescent molecules or particles (quantum dots, nanoclusters, etc.). Such molecules or particles exhibit discrete energy levels. Transitions of electrons in such energy levels lead to excitation and emission of fluorescent light. (B) Schematic diagram of the epifluorescence microscope. In this set-up, illumination and detection of fluorophores is done through same objective.

time spent by an excited fluorophore before emission of the photon and return to the ground state, (iii) biocompatibility, any fluorophore is biocompatible if it allows genuine biological observations without interfering its natural behavior [13,17,19], and (iv) possibility of photobleaching, many of the fluorophores diminish their fluorescence activity upon long-term exposure to excitation light. Mostly organic fluorophores cannot exhibit fluorescence upon long exposure [11]. Quantum dots also exhibit faster or slower photobleaching depending on their environment [20]. Because of ease of observations and availability of resources for fluorescence microscopy, fluorescence imaging techniques are popular. General setup of fluorescence microscope is shown in Fig. 13.1B. It consists of an intense excitation source (usually mercury lamp) and a sensitive photodetector or an imaging device, usually a CCD (charge coupled device) camera for the observation of light emitted by fluorophores. In regular fluorescence microscopes, imaging resolution is limited by the optical diffraction limit. Dependence of diffraction limit on the wavelength used for imaging can be explained by Rayleigh criterion of spatial resolution which depends on numerical aperture parameter explained with Fig. 13.2. R=

1.22l NA condensor + NA objective

Here λ is the wavelength of light used for observation and NA is numerical ­aperture given by é æ D öù D NA i = n sin q = n sin êarctan ç ÷ú » n 2 2 f f è øû ë

13.2 ­ Imaging nanodevices

f

F

q

D

FIG. 13.2 Ray diagram explaining numerical aperture of a thin lens.

where n is the refractive index of the medium in which observations are performed and θ is the maximum of the half-angle of the cone of light that can be collected by objective lens of microscope. Limitations due to spatial resolution can be overcome by advanced fluorescence imaging techniques such as confocal fluorescence microscopy [21], total internal reflection fluorescence imaging [22], or stimulated emission depletion (STED) microscopy [23]. Among these techniques, confocal microscopy set-up is most popular. Basic confocal microscopy set-up consists of an arrangement for pin-hole illumination and collection as shown in Fig. 13.3. To provide sharp monochromatic and intense excitation, laser excitation sources are used in confocal microscopes. In such set-ups the light emitted by fluorophores from the planes other than imaging plane can be eliminated and better imaging resolution as well as confocality can be achieved. Due to these benefits of confocal microscopes, it becomes Light source

Aperture

Detector

Beam splitter

Aperture Focal plane

FIG. 13.3 Schematic diagram of confocal microscope. It can be seen that light from out of focus scatterers or fluorophores can be eliminated using aperture-based set-up.

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easy to capture images from different focusing depths and construct 3D images of the samples. This helps in localization of fluorophores with high resolution in intracellular compartments. Recently developed noble metal quantum clusters (NMQCs) are promising biolabels for confocal fluorescence imaging. We have discussed two major candidates of NMQC family below.

­Monolayer-protected NMQCs Quantum clusters (QCs) are ultrasmall nanoparticles with dimensions in the subnano meter regime and composed of only few tens of atoms [24]. Due to ultrasmall size, QCs do not exhibit plasmonic properties like metallic nanoparticles but due to quantum confinement they possess discrete energy levels and hence photoluminescent properties [25]. Number of atoms in clusters can be tuned by various synthetic methodologies that allow control of their excitation and emission properties [26]. Typical synthetic route for the synthesis of QCs involve reduction of noble metal ions using NaBH4 or other suitable reducing agents [27]. QC core can be stabilized by carrying out reduction of metal ions in the presence of capping agents. For monolayer-protected QCs, capping agents such as GSH (glutathione) [27], BBSH (4-tetra butyl benzene mercaptane) [28], PET (phenyl ethane thiol) [28], CD (cyclodextrin) [29], and SePh (benzeneselenol) [30] have been used in the past. Major properties of monolayer-protected QCs are dependent on the functional groups present on their capping molecules as well as core composition. Various functional groups present on QC’s surface also facilitate conjugation of targeting receptors and drug molecules. Biocompatibility of such monolayer-protected QCs have been demonstrated in the past as ultrasmall size facilitates their clearance through renal route [31].

­Protein-protected NMQCs Unlike monolayer-protected QCs, which are stabilized by small peptide molecules, protein-protected NMQCs are stabilized by bulky proteins [16]. Protein encapsulation not only improves stability of NMQCs in biological environment but also helps in designing an integrated nanodevice, which possess QC properties and protein functionalities at the same time [32]. A variety of proteins have been utilized for the synthesis of protein-protected NMQCs. This involves bovine serum albumin (BSA) [33], hemoglobin (Hb) [34], lactoferrin (Lf) [35], lysozyme (Lys) [36], etc. Most popular synthetic route was reported by Xie et al. in 2009 [33], according to which proteins can be used for the reduction as well as stabilization of QC core. In brief this route involves, incubation of noble metal ions with protein in a basic solution at an appropriate ratio [37]. This route can be regarded as green synthetic route and enhances biocompatibility of protein-protected clusters and hence their applicability for bioimaging. Another reason for using such clusters is that FRET (fluorescence resonance energy transfer) from fluorescent protein capping to QC core can enhance quantum yield and larger signal-to-noise ratio can be achieved for imaging applications [35].

13.2 ­ Imaging nanodevices

­Plasmonic nanoparticles

Due to oxidation and corrosion-resistive properties of noble metals, NMNPs are useful in variety of applications. Among the list of various noble metals present in nature, gold and silver are of biological importance due to their intrinsic properties such as biocompatibility of gold nanoparticles [19a] or antibacterial properties of silver nanoparticles [38]. Also, highly specific LSPR scattering wavelengths and surface-enhanced Raman scattering (SERS) properties of gold and silver nanoparticles have been found useful in variety of bioimaging applications [39]. Such plasmonic NMNPs are usually synthesized in the size regime of 10–100 nm. Multitude of different synthetic routes are available for NMNP synthesis. One of the most popular route was reported by Turkevich et  al. in which gold and silver ions are reduced using trisodium citrate, which also acts as a capping agent to stabilize nanoparticles [40]. NMNPs can be synthesized in different shapes such as spheres, prisms, rods, etc. [41]. Use of miceller templates such as cetyltrimethylammonium bromide (CTAB) helps in control of NMNP growth in such reactions. Different shapes give rise to different LSPR properties that can be used to selectively label intracellular components. Due to biocompatible properties of gold nanoparticles, they are popular as nano-bio labels. Plasmonic nano-bio labels are based on localized surface plasmon resonance (LSPR) property of noble metal nanoparticles (NMNPs). Due to the high electron density of noble metals such as gold and silver, when such NMNPs are exposed to electromagnetic radiation, collective electron charge cloud oscillations exhibit resonance at specific wavelength (Fig.  13.4A). LSPR wavelength of NMNPs depends on the size (Fig. 13.4B) and dielectric properties of their metal constituents. LSPR of NMNPs give rise to enhanced electric field, localized near NMNPs and far field scattering at resonance wavelength [42]. When NMNPs are observed through dark

e- e

e- eMetallic nanopartic nanoparticle

Increase in size

Scattering intensity

Electromagnetic wave

400

(A)

(B)

500

600

700

Wavelength (nm)

FIG. 13.4 (A) Cartoon representation of collective electron oscillations in plasmonic particles upon exposure to electromagnetic waves of specific wavelength. (B) Cartoon representation of the size dependence of plasmon resonance. Increase in size results into red shift in the plasmonic scattering spectrum of nanoparticles.

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Scattering at wavelength based on plasmon resonance of nanoparticle

400

700 1000 Wavelength (nm)

Dark-field objective Nanoparticle

Dark-field condenser

Scattering intensity (counts)

282

White light illumination

400

700 1000 Wavelength (nm)

FIG. 13.5 Schematic of dark field microscope.

field microscopy, scattering provides high signal-to-noise ratio required for imaging [14]. In typical dark field microscopy set-up used for imaging NMNPs, dark field condenser is used for oblique illumination of samples, as shown in Fig. 13.5. When white light is used for illumination in such set-ups, only light of wavelengths matching to LSPR is scattered by NMNPs. This scattered light is then detected by high magnification objective with iris to control and eliminate illumination. A high-sensitivity CCD camera can be used to capture scattering based images of the sample or signal can be routed to the spectrometer, which scans imaging area point by point (or line by line in case of advanced line spectrometers) and provides spectral image of nanoparticles. This allows spectroscopic characterization of NMNPs in biological environments at single particle level [14,15]. Although observations through dark field microscopy are diffraction limited, NMNPs are not sensitive to long-term exposure to illumination light and do not suffer from photobleaching. Hence, NMNPs act as good nano-bio labels for long-term, real-time observations of nano-bio interactions or kinetics of biomolecules inside single cells.

13.3 ­ Nanodevices for the separation of biomolecules and cells

13.3 ­NANODEVICES FOR THE SEPARATION OF BIOMOLECULES AND CELLS 13.3.1 ­ INTRODUCTION During the development of nano-DDS at various stages, it is necessary to separate/ purify the molecules or nanoparticles of interest [43]. These nanodevices can bind to target molecules and then depending on their physical or chemical properties, they can be separated using advanced separation techniques such as fluorescenceactivated sorting, magnetic separation, or using size and charge-based separation columns [2f,44]. Major requirement in such nano-separation devices is that they must facilitate binding of the molecules of interest directly on their surface [45]. For this, NPs can be coated with antibodies against antigens or molecules of interest. Separation approaches can be combined with novel microfluidic devices to separate molecules bound to NPs from small quantity of samples [46]. We have discussed about microfluidic devices for medical nanotechnology applications in a dedicated section in this chapter. We discuss some of the major nanoseparation devices in the following sections.

13.3.2 ­ SIZE- AND CHARGE-BASED SEPARATION (NANO-BASED) This methodology uses either centrifugation technique to separate targeted moleculebound nanoparticles or electrophoretic techniques for charge-based separation of NPs [44a,47]. In case if separability has to be used for the separation of other inseparable molecules (e.g., molecules of same size but different functionality) it is necessary to modify surface of the nanoparticles by targeting moiety or highly charged molecules on NP surface [48]. Targeting moieties or charge on NP surface can attract molecule of interest, which can be later purified by centrifugation or elution from gel slab used for electrophoresis. Separation of charged polymer-coated gold and silver nanoparticles using agarose gel electrophoresis has been demonstrated in the past [43b]. In such samples, size- and shape-dependent separation can be achieved, which can be further useful in understanding size and shape-based interaction of NPs with biomolecules. Separation of NPs with different LSPR can be monitored by simple bright field optical imaging. A model based on the Henry formula has been reported by Hanauer et al. and it can be used to predict gel mobilities of charged polymercoated nanoparticles [43b].

13.3.3 ­ MAGNETIC NANOPARTICLES FOR SEPARATION AND OTHER APPLICATIONS For magnetic separation of biomolecules or cells (bacteria or mammalian cells), samples are treated with antibody functionalized magnetic nanoparticles and then separated using strong magnets [48,49]. Magnetic nanoparticles are usually made up of magnetic materials such as iron, cobalt, or nickel and allow manipulation using magnetic fields [50]. Magnetic nanoparticles are also called as magnetic nanobeads

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and synthesized with size in the range of 5–500 nm or microbeads with size in the regime of 0.5–500 μm. Different types of MNPs have been reported in the past. Among the different types of MNPs, iron oxide nanoparticles are most popular MNPs and also called as Ferrite nanoparticles. These particles possess crystal structure of magnetite or maghemite and can be stabilized by surfactants such as silica, silicone, or phosphoric acid derivatives. Iron oxide nanoparticles with size around 100 nm are superparamagnetic in nature and exhibit magnetic behavior only in the presence of external magnetic field. These particles are also called as superparamagnetic iron oxide nanoparticles (SPIONs). Apart from separation of molecules, SPIONs have been proven as an important tool to enhance magnetic resonance imaging (MRI) contrast, which enables monitoring of anatomical and physicochemical changes in vivo [51]. Although surfaces of SPIONs cannot be easily functionalized by covalent bonding, they can be coated with silica. After silica coating, these particles can be treated with APTES ((3-aminopropyl)triethoxysilane), MPTMS ((3-mercaptopropyl)trimethoxysilane), etc. [52], to develop desired functional groups on their surface. This helps in the conjugation of SPIONs to various targeting molecules for the separation and labeling of biomolecules or cells. Silica-coated SPIONS with narrow size distribution can be obtained, which have benefits of being chemically stable, do not agglomerate, and possess tunable magnetic moment (based on nanoparticle cluster size) [53]. Controlled clustering of such superparamagnetic nanoparticles can help in increasing the magnetic moment and tuning of other properties such as T2 relaxation time important in MRI imaging [54]. Other reported MNPs are metallic MNPs, which provides benefit of smaller size as compared to oxide MNPs for the same magnetic moment [55]. But due to their high reactivity to oxidizing agents, they are not suitable for biomedical applications. Cobalt nanoparticles with graphene shell also have been reported [56]. Such Co-graphene nanoparticles possess higher magnetization properties and are resistant to acidic and basic pH.

13.3.4 ­FLOW CYTOMETERS Cytometry involves analysis of cell suspensions by passing the same through optical excitation and detection set-up [57]. Cytometer can sort labeled or unlabeled cells as well. Depending on the optical signals obtained from single cells, thousands of cells can be analyzed using advanced instrumentation and then cells with specific optical properties can be separated from the sample. Schematic shown in Fig. 13.6 explains the principle of flow cytometer. Briefly, flow cytometers consist of a flow cell, in which cells are aligned and carried as a stream of solvent through sheath fluid. Sheath flow is arranged such that cells are separated by distance larger than their diameter. Stream of cells can be further separated into individual droplets by vibrating mechanism. Aligned cells are then passed through light beam, which excites fluorescent or other optical labels attached to cells or simply illuminate unlabeled cells and then scattered light signal is detected and analyzed. When flow cytometer is used for the separation of fluorescently labeled cells, overall method is called as fluorescence activate cell sorting

13.3 ­ Nanodevices for the separation of biomolecules and cells

Data processing unit Detector

Catcher tube in path

FSC

SSC Fluorescent/granularity 90o

Detector

Sample Sheath fluid Laser Catcher tube outside path

FIG. 13.6 Schematic diagram of the set-up for fluorescence-activated cell sorting using mechanical catcher tube.

(FACS). Using FACS technique, cells can be sorted from a heterogeneous mixture based on their fluorescent signal or scattering properties. Depending on the excitation wavelength and power requirement of the system, mercury or xenon lamps or lasers can be used as excitation light source. Scattered light is detected by detectors in two directions, forward-scattered light (FSC) and side-scattered light (SSC). For cells labeled with fluorescent labels, emitted light needs to be passed through optical filter, which eliminates scattered excitation light before detection. For weaker signals, amplifiers are used to enhance the detector signal before data processing by computer. Recent developments in advanced instrumentation have allowed usage of multiple lasers and detectors in the same system. This allows multiple labeling of cells for phenotyping [58]. Data analysis is an important part of flow cytometry [59]. Measurements obtained from flow cytometer can be either plotted as histograms or scatter plots in two or three dimensions. Regions in scatter plots can be separated by gating for differences in fluorescence intensity, scattering behavior, etc. Gating allows cells with specific scattering or fluorescence properties to be separated from all the cells under flow cytometric analysis. Gating plays major role in cell sorting. Once the data acquisition plot is obtained, gating parameters are identified for the cells of interest and fed into the software. As the cell stream passes, detector gating parameters can be used for real time separation of cells. There are multiple methods available for cell sorting. One of the methods involves use of a mechanical catcher tube to separate cell (Fig. 13.6) [60]. Fig. 13.6

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shows the resting position of catcher tube in sheath flow. Catcher tube is placed at the top end of flow cell. Fig. 13.6 also shows triggered catcher tube inserted in cell stream path to capture a target cell. Catcher tube can be inserted and pulled out of flow to capture the cells. The frequency of catcher tube to insert in and out of flow can be up to 300 Hz. As soon as gating parameters are satisfied by signals from various detectors, a trigger is generated by controller to insert the catcher tube inside flow to capture the target cell. For a fixed flow rate and distance from detector to catcher tube, time required by target cell to reach catcher tube can be calculated and can be used to determine time at which catcher tube can be inserted into flow. Other method for cell sorting involves isolation of target cell by generating vibrations in entire cell stream. Vibrations of sample stream along its axis can be used to break the same into drops. Fig. 13.7 shows schematics of such set-up. For fixed sheath flow velocity and vibrations of the nozzle tip, formation of drops can be obtained with fixed pattern as well as individual target cells can be separated by optimized distance. A voltage charge can be applied to drops containing target cell qualified by predefined gating characteristics and then isolation of cells can be achieved by passing a vibrating stream between positively and negatively charged deflection plates. As the charged drops containing target cells pass through deflection plates, depending on their charge polarity, cells get sorted into separate collection tubes.

Data processing unit Detector FSC

Charging electrode

Fluorescent or granularity Detector SSC

90°

Waste Sorted cells Sample

Deflection plates

Sheath fluid Laser

FIG. 13.7 Schematic diagram of the set-up for fluorescence-activated cell sorting using charging electrode and deflection plates.

13.4 ­ Nano-mems

13.4 ­NANO-MEMS 13.4.1 ­ INTRODUCTION TO MEMS Due to the need for miniaturized sensors and devices at various levels in biomedical applications, microelectromechanical systems (MEMS) have been developed [61]. MEMS devices combine technology of microscopically moving parts with electronics. When such devices are fabricated at nanoscale, they are called as nanoelectromechanical devices (NEMS). MEMS devices can have several interacting components with size of individual component ranging from 10 to 100 μm. These interacting components can be controlled and connected through a central unit, which processes data. Altogether, the size of MEMS device can be from 20 to 1000 μm. While designing MEMS devices, special attention is given to get rid of noise produced by environmental conditions such as electromagnetic interference, vibrations, etc. so as to improve their efficiency in different working environments [62]. Although MEMS are popular as sensing devices, recent advances in biomedical technology are progressing toward the development of new microdrug delivery devices [63]. Such MEMS devices can be used for in vivo administration of drugs or manipulation of internal body organs at microscale, etc. We discuss few examples of such devices and their fabrication techniques in the subsequent sections. Along with MEMS devices, it is often necessary to combine them with microfluidic devices so as to manipulate fluid flows at ultralow volumes. We have discussed about microfluidic devices in a separate section.

13.4.2 ­ FABRICATION OF MEMS Applicability of MEMS in real-life applications has become possible due to advancement in the field of semiconductor device fabrication technologies [62b]. Basic processes developed for the fabrication of electronic devices have been adapted for MEMS fabrication. These processes involve various types of deposition processes, patterning methods, etching processes, sputtering methods, die preparation, etc. Some of the basic devices developed using MEMS are resonators required for biomedical sensing or micropumps for drug delivery, etc. [64]. Major manufacturing techniques used for MEMS devices are discussed below.

­Bulk micromachining

Bulk micromachining is one of the oldest and well-developed techniques for MEMS fabrication [62b,63a,65]. Fabrication of devices using bulk micromachining involves use of single crystal silicon wafer in which whole thickness of wafer is used for step-by-step building of MEMS devices [66]. A variety of etching processes can be used to machine silicon. KOH-etching techniques were developed for bulk silicon fabrication. However, it is difficult to obtain precise structures using KOH-etching technique. To overcome this, novel techniques such as deep reactive ion etching (DRIE) has been developed. Along with etching, anodic-bonding technique can be used to add features of additional silicon wafers or glass plates during m ­ anufacturing

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of MEMS devices [67]. A major benefit of bonding technique is that it does not require any adhesion material to form the bond; for example, two silicon wafers can be bonded together by high-temperature processing in thermal-bonding technique.

­Surface micromachining

Other manufacturing techniques involve deposition of various layers on substrate as the structural materials. Surface micromachining was mainly developed for the fabrication of integrated circuits, which can be combined with MEMS on same silicon substrate. Surface micromachining was used in the development of movable mechanical structures of polycrystalline silicon patterned on underlying oxide layer [68]. Such movable structures can be released by sacrificial etching of the oxide layer and have been useful in the development of accelerometers.

­High aspect ratio silicon micromachining

Techniques such as DRIE (deep reactive-ion etching) can combine benefits of bulk micromachining and surface micromachining. Using DRIE, it is possible to generate MEMS devices, which need comb-like structures with in-plane operation. Using high aspect ratio (HAR) silicon micromachining structural layer thickness of 10– 100 μm is achievable [69]. Epi-poly (polycrystalline silicon) and bonded SOI (silicon on insulator) wafers are commonly used for HAR silicon micromachining.

13.4.3 ­MEMS AND DRUG DELIVERY Although oral delivery of drugs is one of the most prominent methods of drug delivery, MEMS-based drug delivery systems are being developed for special requirements in the delivery of hormones, vaccines, and anticancer agents. Other drug delivery systems such as inhalers, subcutaneous injections, intravenous administration, and infusion pumps suffer from limitations such as poor bioavailability, nonspecific site delivery, lack of monitoring and poorly controlled drug-release profile, etc. [63b]. Drug delivery MEMS devices are basically millimeter-sized reservoirs made using semiconductor fabrication technology. Such drug delivery MEMS consist of tiny channels with microscale caps so that the flow of drug can be controlled [70]. Major considerations while developing new MEMS DDS are, effective drug delivery, ease of use and maintenance, lower cost, lower side-effects and patient comfort [63a]. Automated MEMS DDS can allow replacement of hundreds of injections by single implant procedure. MEMS-based DDS can provide novel solutions for controlled and targeted drug delivery using components such as micropumps [71]. Micropumps offer efficient drug delivery by getting rid of absorption in GIT (Gastrointestinal tract), and degradation in lever. Such micropumps are generally low flow rate (L/ min) piezo actuated diaphragm pumps with much lower power consumption and size as compared to conventional syringe pumps. Automated MEMS DDS can monitor in vivo parameters, such as glucose levels, and take decisions based on preprogramed conditions to release insulin or other drugs [72]. This helps in monitoring spikes in blood parameters and immediate treatment by drug release to avoid any organ

13.5 ­ Microfluidic devices

damage or adverse effects on body functions. For example, in case of osteoporosis treatment, parathyroid hormone is an approved drug that helps the body to repair damage. But parathyroid hormone cannot be delivered by gradual release because it may promote degradation of bones. Hence it needs to be provided by daily injections, which can be achieved by MEMS technology by providing spikes of drug automatically [73]. Multiple hurdles need to be cleared to achieve successful working of MEMS DDS device. Protection of drug until their release is one such challenge. Development of suitable fabrication and packaging technique is necessary to maintain the activity of drug in biological environment. For MEMS DDS components, which degrade over time, MEMS array technology can be used [74]. By MEMS array technology hundreds of sensors or MEMS components can be fabricated in a compact chip and only one sensor/component can be activated at a time and hence increases the life of MEMS implant. MEMS devices can process data on chip as well as transmit it via radio to external computer or controller.

13.5 ­MICROFLUIDIC DEVICES 13.5.1 ­ INTRODUCTION TO MICROFLUIDIC DEVICES Microfluidic devices allow actuation and manipulation of biological or chemical fluids at microscale and tiny volumes. Lab-on-chip microfluidic devices can be used for the development of various bench-top instruments for chemical and biological analysis [75]. This not only reduces the volume of fluids required for analysis but helps in the development of portable instruments for field use. In microfluidic devices it is much easier to control biological environment as compared to large-scale equipment in laboratory. Reduction in the cost and time required for analysis are additional benefits. Range of biological applications such as various biosensors, bacteria/cell detection, and manipulation systems use microfluidic technology. Apart from this, targeted and controlled delivery of advanced therapeutics can also be achieved using microfluidic devices. Microfluidic devices can mimic biological environment by reducing the diffusion times as most of the physicochemical transport processes occur at microscale or nanoscale in cellular environment. Also due to larger surface area-to-volume ratio of microfluidic devices, enhanced mass and heat transfer can be achieved. This can help in exploring microscale interfacial phenomena. Advanced microfluidic devices offer the benefit of easy automation and multimodule processing and analysis required laboratory protocols.

13.5.2 ­ FABRICATION TECHNIQUES FOR MICROFLUIDIC DEVICES Most of the microfluidic devices are fabricated using a polymer called PDMS (polydimethylsiloxane) [75]. Microfluidic devices fabricated using PDMS are flexible, transparent, and much cheaper than silicon or glass devices. Flexibility of PDMS devices also enable fabrication of components such as pneumatic valves,

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which ­cannot be made using rigid materials. Here we will discuss the use of PDMS for the fabrication of microfluidic systems. PDMS is optically transparent, flexible, and nontoxic material with good electrical and thermally insulating properties. These properties of PDMS make it suitable for numerous applications in biology as well as various analytical applications. Microfluidic devices made up of PDMS are fabricated using a technique called as soft lithography [76]. Process flow of soft lithography is shown in Fig. 13.8. Soft lithography uses a patterned master on silicon wafer, which can be replicated into soft elastomers such as PDMS. Due to ease of fabrication steps, which can be carried out in ambient conditions, soft lithography enables simple, inexpensive, and cheaper fabrication. Using a single master on silicon wafer, replication can be repeated several times. Briefly, microfluidic channels can be designed using computeraided designing (CAD) program and converted into a transparency or chrome mask depending on requirements. Such masks are printed in high resolution (~5000 dpi). For durable operation, chrome masks are preferred over transparencies. However,

UV light (i) UV photolithography

Si

High-resolution mask Photoresist

Master

(ii) Replication by pouring PDMS over master followed by cure at 70°C for 1 h

PDMS

(iii) Peel PDMS from master

PDMS

(iv) Bonding on glass

PDMS

Microchannel Glass

FIG. 13.8 Process flow of soft lithography for the fabrication of PDMS microchannels on glass substrate.

13.6 ­ Interfacing nanoelectronics with biology

printing of chrome masks is much costlier as compared to transparencies. These photomasks are then used for 1:1 contact UV photolithography using SU 8 as photoresist to produce a master. Master consists of topographically defined patterns of photoresist on a silicon wafer, which can be used as a mold for PDMS. Generally, a mixture of 10:1 silicon elastomer and a curing agent are mixed and kept in vacuum to get rid of bubbles. Ratio of PDMS and curing agent can be changed to control the flexibility of device. Then, this mixture is poured on to the master and cured for 1 h at 70°C in vacuum oven. After curing, PDMS replica can be peeled from the master, which leads to formation of channels on the PDMS. PDMS replica is then sealed on to plasma cleaned glass, silicon PDMS surface to form channels. Advanced version of soft lithography, capillary molding uses a combination of nanoimprint elastomeric mold. In this method, patterned PDMS mold is kept on the polymer and heated above its glass transition temperature to generate negative replica of the mold. Pattern formation can also be achieved by using a UV curable resin by exposure to UV light. Capillary molding can be used for sub-100-nm lithography using UV curable molds made up of polyurethane functionalized with acrylate groups [77]. These are especially useful in studies related to cell biology.

13.5.3 ­ MICROFLUIDIC DEVICES FOR DRUG DELIVERY Delivery of drugs by oral route is the most prominent route of drug delivery but it cannot work for some of the drug molecules that need to be administered through special routes such as transdermal or pulmonary route. Entry of xenobiotics through any route is prevented by human physiology. Human skin is impermeable to bacteria, large molecules, or other particulates in the surrounding. Respiratory system also has well-developed traps, which can stop entry of aerosolized materials into the lungs. To overcome this, microfluidic devices provide novel routes of drug delivery. Microfluidic devices allow efficient delivery of drugs with portable size of device. Such devices are implantable and consist of microfabricated chambers that facilitate drug-release in  vivo at regular intervals [78]. Such devices can consist of biodegradable micro or nanosized particles, which can encapsulate the drug or therapeutic molecules with the ability to hydrolyze after release in vivo. Implantable microfluidic devices can be made to respond to external (ultrasound or microwave) or environmental stimuli for the release of drugs [79]. Smart devices that respond to environmental stimuli can be integrated with sensors to monitor and analyze physiological signals for the regulation of drug delivery.

13.6 ­ INTERFACING NANOELECTRONICS WITH BIOLOGY 13.6.1 ­ INTRODUCTION TO NANOELECTRONIC DEVICES Basis of biological activities lies in the dynamic actions taking place between molecular machineries at nanoscale. Also due to ultrasmall quantities of biological analytes, it is necessary to develop sensors that can directly interact with biology and

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provide highly sensitive detection of biological signals as well as high-­resolution spatiotemporal monitoring of the same [80]. To solve such issues, an emerging area of nanoelectronics is providing novel and efficient solutions. Such nano-­ bioelectronic interfaces consist of nanostructured surfaces, typically of semiconducting materials, which are highly sensitive to perturbations in electrical signals generated by biological processes. Nanostructured materials can interact efficiently with biomolecules of similar dimensions. Hence, nanoelectronic interfaces can provide highly responsive and rapid sensing devices, which can overcome the limit of detection provided by conventional sensors [81]. Also due to intrinsic properties, nanoelectronic components are easy to integrate with on-chip technologies for easy handling and readout. Advances in semiconductor fabrication technologies, has made it possible to fabricate such sensors in arrays to enable detection of multiple analytes at the same time. Materials used for preparation of nanostructured surfaces are carbon nanotubes, graphene sheets, or other rod/nanowire-like materials. Uses of different materials enable different sensing abilities and physicochemical interactions with biology, which is useful in making variety of device configurations. Choices of materials for particular application are based on responsiveness of the same to analyte at nanodimensions, suitable fabrication methods, cost, etc.

13.6.2 ­ APPLICATIONS OF NANOELECTRONICS IN BIOLOGY AND DRUG DELIVERY Nanoelectronic sensors have defied ultimate limits of detection. For example, transistors made of SWCNT (single-walled carbon nanotube) and SiNW (silicon nanowires) have been used for single-molecule and single-virus detection, respectively [81a,82]. Nanoelectronic sensors have also been used for detection of signaling molecules released by live cells or to detect bioelectrical signals by neuronal cells [83]. One of the most prominent work done by Charles Lieber’s group has demonstrated the use of nanowire transistors for spatially resolved studies of nerve impulses by individual neurites [84]. Such approaches are especially useful due to their noninvasive methodology, higher sensitivity, and resolution as compared to conventional microelectrode techniques. Even for invasive experiments, nanoelectronic components offer ways to measure desired signals from single cells by minimal invasive probes. Arrays of nanoelectronic sensors can be used for high-resolution mapping of signals in cellular network of brain or heart tissues. Such studies are useful in understanding the effect of targeted drugs on neuronal or heart cell functions, hence useful for diagnosis and drug discovery. To improve the applicability of such nano-bioelectronic methods, it is necessary to develop easy and scalable methods for the fabrication of nanodevices. It is also necessary to understand nano-bio interactions in detail so that crucial modifications required in the nanoelectronic tools can be identified.

13.7 ­ Nanorobots

13.7 ­NANOROBOTS 13.7.1 ­ INTRODUCTION TO NANOROBOTS So far, nanorobots are theoretical devices composed of nanocomponents with dimensions in the range of nanometers (10−9 m). Nanorobots are designed and built for controlled manipulations at molecular or cellular level [85]. Nanorobots are different from microrobots as the scale and materials used for construction are different but conceptually designed, and control of both can have similarities. Advances in fabrication technologies have made it possible to realize nanorobots, which can be used for the manipulation of microscopic world. Biomimetics is one of the easiest ways to design and build efficient nanorobots. Carbon can be considered as major element to construct nanorobots for medical applications [86]. Carbon can be in the form of different types of nanocomposites to build nanorobots. Other elements like hydrogen nitrogen, oxygen, sulfur, silicon, etc. can be used to make special components of nanorobots like nanoscale gears. Nanorobots can be made to respond to environmental stimuli or external controlling signals so that they can reach their target inside human body and perform assigned tasks at single-cell level.

13.7.2 ­ NANOROBOTS FOR CELL REPAIRING AND DRUG DELIVERY Although nanomachines for cell repairing and drug delivery are largely in hypothetical stage, some of the preliminary models have been tested. For example, singlemolecule car, which can be actuated by changing environmental temperature and by positioning a STM (scanning tunneling microscope) tip have been demonstrated [87]. Nanomachines have been designed to measure concentrations of chemical components in cellular environments or to detect and destroy cancer cells, etc. Nanorobots can be built to perform variety of desired tasks in biomedical or other technological fields. In science fiction, it has been always imagined that nanorobot can be made such that they can replicate themselves and work in groups to perform macroscopic tasks. Nanorobots designed to work inside human body can be constructed using components, which are inert to biological environment inside human body. For example, exterior of nanorobots can be made using diamondoid structure of carbon atoms, which possess higher strength but inert to environment [85b]. Such unreactive materials help in reducing the risk of triggering immune system and nanorobots can perform their task smoothly. The smoother and more flawless the diamond surface, the more helpful it is in reducing the leukocyte activity [88]. When exterior of devices can be made with smoothness of nanometer precision, it exhibits very low bioactivity. Chemical components from blood, such as glucose, oxygen can be used to energize nanorobots. With their ultrasmall dimensions they can traverse through blood vessels. To permit the passage of nanorobots through blood capillary, size of nanorobots can be in the range of 0.5–3 μm. In future, tabletop instruments can be used to fabricate/replicate nanorobots for patient-specific needs. Such nanorobots can be then injected into human body to achieve quick and painless elimination of diseases or any other medical conditions

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such as accidental damage to organs. In the context of cancer treatment, medical nanorobots can be helpful in targeted treatment and elimination of tumors. This will be achieved by constructing nanorobots that can correct genetic defects or recognize and destruct cancer cells, which cannot be achieved by natural immune response of body. Also as shown in many of the science fictions, nanorobots can be used to enhance human capabilities. However, so far nanorobots are fabricated only in very carefully controlled manufacturing environment and they will not be allowed to replicate on themselves inside human body.

13.8 ­SUMMARY To summarize, we have discussed regular as well as cutting-edge medical nanotechnology devices in this chapter. The most basic applications of medical nanotechnology devices involve labeling using fluorescent and plasmonic nanoparticles. Among fluorescent nanoparticles, quantum dots have been investigated by numerous imaging applications but recently developed quantum clusters of noble metals are still in the testing phase. However, plasmonic nanoparticles that are one of the most efficient labeling agents for single particle imaging or tracking are popular and improving in terms of applicability using advanced instrumentation and combining with SERS. Apart from nanoparticles used for labeling, we also discuss use of nanoparticles for the separation of molecules and pathogens for detection. In such applications, magnetic nanoparticles dominate other particles available for separation due to ease in their separation process. We have also discussed separation techniques such as fluorescence-activated cell cytometry and its principles. In cutting-edge development and applications of medical nanotechnology devices, we have discussed microfluidic devices for analysis and its integration with MEMS devices for advanced drug delivery systems. We end this chapter with the most advanced version of MEMS, nanorobots that are still in hypothetical stage but promise bright future, which can change the way drugs are administered and monitored. We believe that this brief introduction of various nanotechnology devices will be useful for beginners in this area.

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