Manipulation of biological samples using micro and nano techniques

Abstract

The constant interest in handling, integrating and understanding biological systems of interest for the biomedical field, the pharmaceutical industry and the biomaterial researchers demand the use of techniques that allow the manipulation of biological samples causing minimal or no damage to their natural structure. Thanks to the advances in micro- and nanofabrication during the last decades several manipulation techniques offer us the possibility to image, characterize and manipulate biological material in a controlled way. Using these techniques the integration of biomaterials with remarkable properties with physical transducers has been possible, giving rise to new and highly sensitive biosensing devices. This article reviews the different techniques available to manipulate and integrate biological materials in a controlled manner either by sliding them along a surface (2-D manipulation), by grapping them and moving them to a new position (3-D manipulation), or by manipulating and relocating them applying external forces. The advantages and drawbacks are mentioned together with examples that reflect the state of the art of manipulation techniques for biological samples (171 references).

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Jaime Castillo

Jaime Castillo received his PhD degree in 2005 at the Department of Biotechnology, Lund University, Sweden. His PhD work involved the fabrication of electrochemical biosensors for the detection of compounds of biomedical importance using cellular models. He currently holds a position as Postdoctoral fellow at the Department of Micro and Nanotechnology, DTU Nanotech, Technical University of Denmark. His research focuses on micro and nanotechnologies for the development of biosensing devices for biomedical applications. A strong focus is currently set on manipulation, characterization and integration of biological nanotubes and nanofibers with micro and nanostructures for the development of bioelectronic devices.

Insight, innovation, integration

Advances in micro and nanofabrication allow the manipulation of biological samples in a controlled way enabling integration with micro and nanostructures. In this way the biological material can be characterized and used to build sensitive and selective sensor devices, while trying to keep the samples in their natural structure and environment. This article presents the state of the art of different techniques used for this purpose. The advantages and disadvantages are discussed in a critical manner. Additionally, several examples are included and described.

Introduction

Manipulation of μm and nm-scale biomaterials and integrating them in different devices has become a critical issue in the bio-nanotechnology field during the last decade.^1–3 The linking of our macroscopic world to the nanoscopic world of single molecules, nanoparticles and functional nanostructures is a technological challenge.⁴ Different techniques have emerged to help scientists overcome the obstacles of size and precision when interacting with tiny objects such as micro and nanoparticles. These techniques could involve the direct interaction between an instrument and the object that needs to be manipulated, as in the case of 2-D and 3-D manipulation, or could involve the interaction of the object with external electric or magnetic fields for finally placing the object in a specific location, such as a microelectrode.

The selection of the right technique to manipulate the biological component will depend on different aspects such as its size and shape or the medium in which we find the component, either dry or wet. Handling nanoscale objects includes finding these objects, tracking and moving them. Two-dimensional nanomanipulation using scanning probe microscopy allows nanocomponents to be transferred along a surface with nm level precision. However, transfer of nanocomponents from a “source substrate” to a “target substrate”, such as picking a nanocomponent from a solution and placing it between two electrodes or mounting it as the extension of a scanning probe tip, is difficult to do without full 3-D monitoring and manipulating capability.

While 3-D manipulation is what we need to transfer biological samples onto a specific location, it is instructive to review the established methods of nanomanipulation. Typically the type of microscope used completely determines the possibilities and the limitations in the case of 2 and 3-D manipulation. For instance, the scanning probe microscopes, scanning tunneling microscopes and atomic force microscopes, offer the best resolution and precision, but in some cases only work in 2-D, while electron microscopes provide more possibilities to manipulate objects in 3-D.⁵

Scheme 1 presents the different strategies for nanomanipulation depending on the environment (air, liquid, or vacuum) in which the sample is and the size of it.

Scheme 1 Microscopes, environments and strategies for nanomanipulation. The different strategies for nanomanipulation are determined by the environment (air, liquid, or vacuum), which is decided by the properties and size of the objects, and observation methods. Reproduced with kind permission from T. Fukuda et al., in IEEE, 2003, 1803–1818. (© [2003] IEEE).⁶

The different nanomanipulation systems can be grouped according to the utilized starting point, manipulator and object interaction process, and operation techniques as shown in Scheme 2, which is a modification of the scheme used by Sitti.⁷

Scheme 2 Grouping of nanomanipulation systems depending on starting point, utilized process, manipulator and object interaction type, and utilized operation technique. Reproduced with kind permission from ref. 7.

Effects of scaling down

One of the big challenges for researchers aiming to manipulate biological micro- or nanostructures is to understand and exploit the changes in behaviour that occur when going down in size. Effects that are negligible at the macroscopic scale become dominant at the micro- and nanometre scale. This situation forces us to modify our way of thinking, by taking into account the new effects that dominate at the micro and nanoscale. An example of this scaling effect can be found in microfluidics. When decreasing the size gravity no longer plays a role in a microchannel, while forces that at the macroscale are insignificant are now dominating, such as surface tension. Another example is the result of scaling of attractive forces; at a radius below 10⁻⁴ m electrostatic forces dominate over gravity, Fig. 1.

Fig. 1 Results of scaling of attractive forces. For r < 1 m; the magnetic force is sufficient to lift the sphere. Below r = 10⁻⁴ m, the electrostatic force dominates over gravity, and for r < 10⁻⁷ m, the van der Waals force is higher than the weight of the sphere. Reproduced with kind permission from J. J. Abbot et al., IEEE Robotics & Automation Mag., 2007, 14, 92–103. (© [2007] IEEE).⁸

It is then necessary to understand the scaling laws in different fields such as mechanics, electromagnetism, fluids, optics, thermodynamics, and quantum effects. This will facilitate the understanding of the transition from the macroscopic to the micro- and nanoscopic dimensions. A complete description of these scaling laws is beyond the scope of this report; there are several reviews that deal with the topic of scaling:^8–13

Manipulation systems in 2D

Manipulation with scanning tunnelling microscope (STM)

The STM is the ancestor of all scanning probe microscopes and was the first instrument to generate real-space images of surfaces with atomic resolution. Scanning probe microscopes are a class of tools that scan a sharp probe tip across a sample and provide nanometre-scale information concerning the sample. The tip force of the scanning probe microscope was used as a manipulation tool by Eigler and Schweizer, who pushed Xenon atoms along a nickel surface in almost absolute zero temperature conditions.¹⁴ This was the first example of nanomanipulation in 2-D using an STM.

In comparison with other techniques such as transmission electron microscopy (TEM) an STM can interact with biological samples without staining or coating the samples with heavy atoms such as Os, Pb, Ur, or Pt resulting in loss of resolution and disruption of the molecular structure. Additionally STM can operate in gaseous and liquid environments, thus avoiding the sample exposure to high vacuum, that can remove water molecules essential to the stability of the sample’s natural structure.¹⁵STM also has the highest spatial resolution amongst scanning probe microscopy techniques. Individual atoms and molecules on a conductive surface can be imaged by STM.¹⁶

An example of the use of STM for manipulation at molecular scale can be found in the work of Griessl and coworkers. They placed an individual football shaped molecule, C60, in the middle of one of the template rings and then “kicked” the molecule into the guest position of the neighbouring ring.¹⁷

Manipulation with atomic force microscope (AFM)

Atomic force microscope based systems enable monitoring of the forces during manipulation, which can provide important information about the interaction between the nanocomponent and the substrate, as well as about intrinsic properties of the component itself. By using AFM it is possible to push, pull, cut, and indent any material and surface in air, liquid or vacuum, Fig. 2. The choice of the phase in which the interaction between the AFM tip and the biomaterial to be analyzed is done will depend on the medium the specimen will find as near lifelike conditions as possible. The great advantage of the AFM for biologists is that it can image and manipulate samples in a fluid environment.¹⁸

Fig. 2 (a) An SEM image of a silicon AFM probe with about a 10 nm tip radius (scale bar: 10 μm) and (b) example 1-D and 2-D mechanical micro/nanomanipulation tasks using an AFM probe. Reproduced with kind permission from M. Sitti, IEEE Robotics & Automation Mag., 2007, 14, 53–60. (© [2007] IEEE).¹⁹

AFM manipulation relies on the tip of the AFM probe both for imaging and for manipulation in separate, alternating steps. The main strategy is as follows: (1) image the surface with semi-fixed nanoobjects using AFM tapping mode for minimal distortion of the particle positions and (2) push the object by the AFM probe tip in contact mode. This type of control scheme is known as “look and move” and requires the use of a scanning electron microscope for the observation of the manipulated object and the AFM probe under real time.¹⁹AFM allows pushing, pulling, cutting and indenting any biological material in any environment as shown in Fig. 2.

Among the other instruments of the scanning probe microscope family the AFM has the widest range of applications, both for imaging and nanomanipulation. However, standard AFM systems are restricted to small working areas and operating speed efficiency is quite limited.⁴ The separation of the imaging and manipulation makes nanomanipulation unnecessarily difficult and clumsy. Since macroscale manipulation is an activity that involves coordination of eye and hand, with simultaneous visual and force feedback needed for efficiency, researchers have tried to transfer these features to scanning probe microscope based manipulation.

In an atomic force microscope, the force can obviously be measured very accurately, and an input device—a sophisticated mouse—with force feedback could allow the operator not just to detect the position of nanoobjects, and push these around, but also to “feel” the action and even see the results in 3-D with virtual reality equipment (stereo display and glasses).²⁰ AFM systems with high resolutions have been coupled with video microscopy and non-contact ablation by ultraviolet (UV) microbeam laser for large scale manipulation of biological specimens.⁴AFM is used to remove layers of material covering the area of analysis of interest by repeated scans of the same area or in a faster way by increasing the force applied. More examples of the use of AFM with biological samples can be found in Table 1.

Table 1 Application of the different manipulation techniques for the manipulation of biological samples

Manipulation technique	Manipulated sample	Ref.
AFM	Human chromosomes	4, 104, 105
AFM	Lactate oxidase	106
AFM	Bovine carbonic anhydrase	107
AFM	Synaptic vesicles	108
AFM	Pseudomonas putida	109
AFM	DNA molecules	110
AFM	DNA/Ppynanowires	111
AFM	Single DNA strands	23
AFM	Microtubules	112
AFM	Virus	113, 114
SEM	Carcinoma cells, fungi	27
SEM	Human erythrocytes	115
SEM	Fungal cells	116
SEM	Bacteria	117
SEM	Osteoclast cells	118
TEM	Cellulose films	119
TEM	DNA	41
Microgripper	HeLa cells	120–122
Microgripper	Porcine aortic valve interstitial cells	123
Microgripper	Cells	124
Self-assembly	Collagen fibrils	26
Self-assembly	Amyloid protein fibres	125
Self-assembly	Protein nanotubes	126
Self-assembly	DNA	23
Self-assembly	Peptide amphiphile fibres	127
Optical tweezers	Au nanoparticles and DNA	128
Optical tweezers	DNA	129
Optical tweezers	Red blood cells, yeast cells	98
Optical tweezers	Viruses and bacteria	80, 130
Optical tweezers	Myosin	82
Opto-electrostatic	Cells and single DNA molecule	131
Optical microscopy	Yeast cell	132
Laser tweezers	Oligodendroglia cells	133
Laser tweezers	Neuroblastoma cells	133
Laser tweezers	Cells	78
Laser tweezers	Murine fibroblast cells	134
Laser tweezers	Sperm cells	135
Laser pulses	Cornea tissue	136
Laser-based microdisection	DNA, RNA, proteins	137
DEP	Peptide nanotubes	64
DEP	Cardiac myocytes	138
DEP	Red blood cells, E. coli	139
DEP	DNA	55, 140–142
DEP	DNA, proteins, viruses, bacteria, chromosomes	143
DEP	Lymphocytes	144
DEP	Insulinoma cells	63
DEP	Jurkat T cells and HL60 leukemia cells	145
DEP	Breast cancer cells	146, 147
DEP	Human leukocytes	148
DEP	MDA-435 human breast cancer cells, HL-60 leukemia cells, DS19 murine erythroleukemia cells, blood cells	149
DEP	MCF10A human breast epithelium cells, MCF7 human breast cancer cells	150
DEP	Yeast cells	151
DEP	Human cells	152
Acoustophoresis	Lymphocytes, red cells, platelets	90
Acoustophoresis	Neural stem cells	91
Acoustophoresis	Erythrocytes	89, 92, 153
Acoustophoresis	HeLa cancer cells	120
Magnetic fields	Amyloid nanotubes	73
Magnetic fields	Endothelial progenitor cells (EPC)	154
Magnetic fields	Single DNA	76
Magnetic fields	Blood cells	155, 156
Magnetic fields	Single yeast cells	157
Magnetic fields	Bacteria	158
Magnetic fields	NIH/3T3 fibroblasts	159
Magnetic fields	DNA structures	77
Magnetic fields	Mouse macrophages	160
Magnetic fields	DNA	75
Magnetic fields	DNA	74
Nanotweezers	DNA	41
Nanotweezers	DNA	35
Microfluidics	Blood cells	101, 161–163
Microfluidics	DNA	100
Microfluidics	Red blood cells, yeast cells	98
Microfluidics	MDA-435 human breast cancer cells, HL-60 leukemia cells, DS19 murine erythroleukemia cells, blood mononuclear cells	149
Microfluidics	Cells, proteins	164
Microfluidics	RecA filaments, single DNA	99
MEMS	Mouse oocytes and embryos	165
MEMS	HeLa cancer cells	120, 121
MEMS	Cabage cells	166
MEMS	Yeast cells	68, 167, 168
MEMS	Single embryo cells	169
MEMS	DNA, protein	102, 170

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Atomic force microscopy allows the study of the mechanical properties of biological structures on the micro- and nano level. Changes in the mechanical properties of single cells reflect their physiological state; this can be used to identify pathological states such as cancer. In this way healthy cells could be differentiated from cancer cells.²¹

AFM also plays an important role on the road to molecular manufacturing.²² Combining the cut-and-paste surface-assembly technique, proposed by Gaub and co-workers,²³ together with parallel AFM cantilevers, will bring us closer to this goal by improving the control of the shape, size and chemical composition of functional structures on surfaces at the molecular level. Still, issues such as slow transfer rate and the reproducibility of the AFM tip picking up individual units need to be improved.

Manipulation in scanning electron microscope (SEM)

In parallel to the usage and the advancement of AFMs for nanomanipulation in the early 1990’s, researchers started to develop positioning systems for the integration into SEMs.²⁴ The scanning electron microscope is a microscope that uses electrons instead of light to form an image. The scanning electron microscope has the advantage of providing a considerably higher resolution than the optical microscope as well as a large depth of field, which allows more of a sample to be in focus at one time. Nowadays SEMs can have a resolving power of 1 nm and can magnify over 400 000 times.

The first reported use of the SEM for manipulation can be found in the nanomanipulator system, called the nanorobot system developed by Hatamura and Morishita at the Tokyo University.²⁵ Their aim was to manipulate micro-objects smaller than 100 μm in order to construct micro-devices.

In 2005 Fahlbusch and co-workers developed a sensor-based manipulation and processing system for nano-scale objects operating inside a SEM (Lab-in-SEM). This laboratory in SEM allows for semi-automated manipulation, processing, and testing of nanocomponents and samples using different tools without the need to open the SEM chamber, thus improving reliability, reproducibility and productivity.²⁴

Several companies are working on systems for manipulation work on a full wafer, and automated manipulation assembly. Commercial systems for SEM manipulation are manufactured by Zyvex (http://www.zyvex.com). This system allows electrical characterization of integrated circuits for failure analysis, micro- and nanoassembly, sample preparation and positioning, basic nanomanipulation and surface science experiments. The Zyvex S100 was used for the manipulation of collagen fibres.²⁶

The ultra-high vacuum and low temperatures required and the limitation of electrically conducting surfaces are the large constraints for using this technique as a manipulation tool for biological samples.²⁴ Biological samples need to be fixed, dehydrated, critical point dried, and coated before they can be observed in a conventional SEM. These procedures frequently produce artifacts, since they alter the size and shape of the samples. These drawbacks can be avoided by using an environmental scanning electron microscope (ESEM). This equipment allows imaging and manipulation of biological samples in their natural state and without metal coating.²⁷ Despite the convenience of ESEM having been demonstrated in many biological applications, many authors reported difficulties when using ESEM in wet samples.^28–30 Based on these reports it can be concluded that ESEM cannot replace SEM, instead they can complement each other in order to provide a real image of the sample and resolve finer structures in detail.³¹

Manipulation in transmission electron microscope (TEM)

To understand how the transmission electron microscope works, we can compare it with a slide projector. In the electron microscope, the light source is replaced by an electron source (a tungsten filament heated in vacuum), the glass lenses are replaced by magnetic lenses and the projection screen is replaced by a fluorescent screen which emits light when struck by electrons. The whole trajectory from source to screen is under vacuum and the sample has to be very thin, usually 0.5 μm or less, to allow the penetration of the electrons. Modern TEMs can resolve 1 nm at magnifications of up to 1 million times.

Very high precision manipulation can be done with a TEM, but the lack of space makes “pick-and-place” assembly of devices extremely difficult and impractical. Additionally, biological molecules are built of atoms such as C and H, which have low atomic numbers with low electron densities, resulting in very low-contrast TEM images. To improve the contrast it is necessary to stain or coat the molecules with heavy atoms, which results in loss of resolution and disruption of the molecular structure. Additionally, biological samples are further disrupted by the high vacuum removing water molecules, which alters the stability of the natural structure. Finally the high-energy electron beam of TEM could cause radiation damages on the samples.¹⁵ Nozawa and co-workers at Tokyo University fabricated nanoneedles with sharp tips with radii in the nanometre range that had big potential to be used to explore biological samples.³² Micromachined tunneling tips integrated with positioning actuators were used to manipulate DNA bundles. The same system was used to manipulate other filamentary biomolecules and is envision to be used with more complex biological systems.^33–35

Nanofactory Instruments offers tools for electrical and mechanical probing inside the TEM. This combination of techniques enables simultaneous and correlated contact probing and manipulation of the sample while observing the sample with the usual TEM techniques (http://www.nanofactory.com).

Manipulation systems in 3D

Microgrippers and nanotweezers

Microgrippers and nanotweezers are devices that use variable force for picking and placing an object. An example of these devices is the thermal gripper that uses heat induced expansion of a material to generate a force.⁵ When compared with tools such as scanning probe microscope tips, a microgripper could provide better control over the applied forces, as well as a more well-defined mechanical grip on the object since rotation will be limited by the gripper geometry. A microgripper also gives the possibility to make direct electrical and mechanical measurements on the grabbed objects if the gripper arms are conducting or can provide a force feedback signal.³⁶

Peter Bøggild’s group at the Technical University of Denmark has been working since 1999 with the fabrication of 3D manipulation tools such as silicon microgrippers, nanotweezers and nanostructured non-stick coatings to avoid adhesive effects in nanomanipulation tasks.^36–38 A series of videos showing their work with microgrippers and nanostructured surfaces can be observed in this address: http://www.youtube.com/profile?user=NanoClips

The nanotube nanotweezers fabricated by Kim and Lieber are one of the most well-know nanomanipulation tool in terms of the gripper size. With this tool they were able to manipulated GaAs nanowires and polystyrene nanoclusters.³⁹ Nanotweezers have also been used for the manipulation of nanowires⁴⁰ and DNA molecules, Fig. 3.^35,41

Fig. 3 (a) Principle of DNA retrieval by AC dielectrophoresis; (b) SEM photograph of a bundle of DNA trapped between silicon nanotweezers fabricated by Yamahata and co workers. Reproduced with kind permission from C. Yamahata et al., in International Conference on Microtechnologies in Medicine and Biology, 2006, 123–126. (© [2006] IEEE).³⁵

Companies like Nascatec (http://www.nascatec.com) produce nanogrippers that can operate in a standard atmosphere as well as in UHV conditions. The Nascatec nanogripper can be fabricated with tweezer openings of 170 μm and 50 μm. Zyvex Instruments (http://www.zyvex.com) fabricated the NanoEffector® microgripper. With this tool it is possible to remove and place samples onto a TEM grid inside a scanning electron microscope (SEM). These grippers use electrothermal actuation to achieve a range of motion from zero to five microns of opening at the tips. An electrical potential is applied across the two anchor pads, passing current through the bent-beam structures. Joule heating within the beams results in localized thermal expansion. This thermal expansion drives flexures which amplify and create horizontal motion at the gripper tips.

One of the big drawbacks of the use of microgrippers with biological samples is the generation of electrical fields or thermal gradients which can destroy the samples. Mechanically actuated grippers have been fabricated to overcome these drawbacks but still the risk of damaging or contamination of the sample due to the contact with the manipulation device.^42,43

Manipulation using electric or magnetic fields

In a society craving for faster, cheaper but also reliable biochemical analysis of patient samples, research has turned towards miniaturisation of existing processes in the so-called lab-on-a-chip systems, where test tubes and huge sample volumes are replaced by microfluidic channels requiring only a minimum sample volume for operation. Handling of these samples (mixing, pumping, and concentrating) requires actuation of fluids and one of the most efficient methods for the purpose is electrokinetics.

Electrokinetics covers a number of different phenomena occurring when an electric field is applied inside a fluid. In the case of direct current (DC) fields the most known effects are those of electrophoresis (EP) and electroosmosis (EO). EP is the movement of a particle due to the effects of an electric field on the net charge of a particle. EO in a channel is the movement of the fluid created by the interaction between the electric field along the channel with the electrical double layer at the interface between an electrolyte and a solid. These two phenomena can be used for particle separation and/or fluid flow; however, they are not without problems. A high voltage is required in order to have visible results, something which in itself is a problem, but which can also leads to the creation of bubbles and the onset of electrochemical reactions, both of which are not wanted in a microfluidic system.

To minimise these problems, AC electrokinetics has been receiving increasing interest in recent years. Whilst DC fields act on the net charge of particles, AC fields act on induced charges. As both the field and the charges simultaneously change polarity at each cycle, the force acting on them will always be in one direction and movement can be generated. There are mainly three types of phenomena associated with AC fields. Dielectrophoresis (DEP), AC electroosmosis and AC electrothermal effects. DEP is the movement of a polarisable particle in an inhomogeneous electric field and was discovered back in the 50s by Pohl.^44,45 It is by far the most commonly used method for the manipulation of biological structures, so we will concentrate on it in the remainder of this section. For completeness, AC electroosmosis is the fluid motion induced by moving charges in the double layer, while AC electrothermal effects refer to fluid motion caused by the interaction of electric fields with conductivity and permittivity gradients in the fluid developed by Joule heating.⁴⁶

Dielectrophoresis

Most of the literature available on DEP on biological structures concentrates on different types of cells and to a lesser extent on DNA. Primary applications of the method involve cell sorting, e.g. live from dead cells,^47,48 healthy from cancer cells,^49–51 sorting between different types of cells,^52–54 size sorting of DNA,⁵⁵ dielectric properties mapping,^55–58 and biological structure trapping,^58–60e.g. in order to create cell clusters.⁶¹

A summary of some of the literature used in this paper is found in Table 1 in terms of authors, method used and biological structure used. To name but a few examples, Wang et al.⁵⁴ used interdigitated electrodes at the bottom of a chamber to enrich leukocytes from blood and separate T-, B-lymphocytes, monocytes and granulocytes. Differentiation of human leukocyte subpopulations is very important e.g. for the analysis of the functionality of the different leukocytes in immune responses.⁵³ As most biological structures survive in rather high conductivity solutions Marcus et al.⁶² have studied how DEP works in such environments using carbon nanofibers. When properly functionalised, these nanowires can work as biologically actuated electrical switches. The authors demonstrate that DEP can work in saline solutions when care is taken to keep the frequency above a certain critical limit. In a recent article Pethig et al.⁶¹ created insulinoma cell clusters of about 1000 cells, which they term pseudo-islets, as the structures resemble in size the islets of Langerhans, found in the pancreas, where they regulate insulin levels in the blood. The authors hope to be able to uncover how cells work together in complexes in the body, Fig. 4.

Recently in our group, the manipulation of biological peptide nanotubes was achieved by DEP. Several and even individual peptide nanotubes were manipulated and immobilized on gold microelectrodes allowing the study of the conductivity of these nanostructures, Fig. 5.⁶⁴

Fig. 4 A computer plot of the square field gradient in the structures used in Pethig et al.⁶³ The location where cells from a cluster under negative DEP is shown. This figure first appeared in IET Nanobiotechnology. Reproduced here with permission. Copyright IET. (© [2008] IEEE).⁶³

Fig. 5 Self-assembled peptide nanotubes manipulated and immobilized on top of gold microelectrodes by using dielectrophoresis. J. Castillo et al., Manipulation of self-assembly amyloid peptide nanotubes by dielectrophoresis. Electrophoresis, 2008, DOI: 10.1002/elps.20080026064. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.⁶⁴

Besides the DEP potential for the manipulation of nanostructures, it also offers important advantage of being compatible with standard microelectronics foundry technologies, allowing the integration of nanodevices with transduction, readout, and signal processing and communications circuitry.

Precision control and in situ manufacturing process monitoring are the two main drawbacks reported for the use of DEP in nanomanipulation processes. These drawbacks have limited the yield and future commercialization of DEP systems.¹⁹

Manipulation using magnetic fields

Magnetic alignment is an extremely versatile technique that exploits the anisotropy in diamagnetic susceptibility of assemblies of molecules. It is a contact free technique, homogeneously effective over the whole sample and can be used to produce either structured thin films as well as bulk material.⁶⁵ Manipulation of biological cells is achieved by magnetic beads that can be attached or engulfed by cells with high selectivity. The magnetic bead can be fucntionalized with antibodies, peptides or lectins to interact with the biological material.⁶⁶ The most commonly used magnetic beads are between 10 and 100 nm in diameter. Their small size make them very gentle on the cells with aparently no damage on the cellular function or cell viability.⁶⁷ External magnets are required to perform the manipulation of the cells containing the magnetic beads.⁶⁸

Biological materials such as helix-turn-helyx peptides ,⁶⁹ lysozyme crystals,⁷⁰ cells,⁷¹ fibrin⁷² and amyloid peptides nanotubes,⁷³ have been manipulated using magnetic alignment. The elastic properties of single DNA molecules were studied by using magnetic traps which allowed pulling and twisting a single molecule. In this study different models were used to describe DNA under tension.^74–76 A practical example of the use of magnetic fields to manipulate biomaterial can be found in the work of Reches and Gazit.⁷³ They aligned fibrous assemblies from low-molecular-weight peptide amphiphiles by the use of a high magnetic field, Fig. 6.

Fig. 6 Self-assembly of peptide nanotubes using an external magnetic field. (a) Schematic representation of the dipeptide monomers self-assembled in the presence of a solution containing magnetite nanoparticles. (b) TEM image of a self-assembled peptide tube coated with magnetic nanoparticles. (c) SEM micrograph of the self-assembled magnetic tubes. (d) Horizontal arrangement of the magnetic peptide tubes after their exposure to a magnetic field. (e) Schematic representation of the self-assembled magnetic nanotubes. (f, g) Scheme of the magnetic nanotubes randomly oriented before exposure to the magnetic field (f) and horizontally aligned on exposure to the magnetic field (g).⁷³ Reprinted by permission from Macmillan Publishers Ltd: Nature Nanotechnol., M. Reches and E. Gazit, Nat. Nanotechnol., 2006, 1, 195–200, copyright 2006.

The principal drawback of this technique is the fact that the aligning force is very small. Because of this alignment will occur only when molecules present in the sample are very large and contain moieties with high magnetic susceptibility, such as aromatic units.⁶⁵ Additionally, the controlled manipulation and movement of individual magnetic beads requires more intricate control over external magnetic fields and despite efforts in the last decades to optimize magnetophoretic conditions for this purpose, applications involving magnetophoresis for manipulation of biological samples are still scarce.⁷⁷

Manipulation using optical tweezers

Optical trapping occurs due to a gradient force on a particle in an optical field. As a result small particles such a micro spheres, micro beads or biological cells can be manipulated and confined to small regions, Fig. 7.⁷⁸ Optical tweezers are a non contact manipulator tool that apply trapping forces on the order of piconewtons with resolutions as fine as 100 aN.¹⁹ These values are ideal for exerting forces on biological samples and macromolecular sytems.⁷⁹ Ashkin and co-workers at the AT&T Bell Laboratories described for the first time the optical trapping of dielectric particles. In this study a spherical Mie particle was trapped in water by the highly convergent light of a single-beam gradient force trap. Their results suggested the use of single-beam force traps with biological particles such as viruses.^80,81 Laser tweezers have been used extensively in biological systems for in vivo manipulation of cells and single molecule studies. Biological motors, DNA, RNA, viruses, chromatin fibres, and other biomaterials can be manipulated in almost 3-D in their physiological liquid conditions by focusing a laser beam to a point. Forces in DNA transcription and packaging were investigated using optical tweezers by Bustamante and co-workers.⁷⁴ Optical tweezers were used in this study to apply forces on DNA and in this way make direct measurements of its mechanical properties.

Fig. 7 Optical tweezers use a strongly focused beam of light to trap objects. Intensity gradients in the converging beam draw small objects, such as a colloidal particle, toward the focus, whereas the radiation pressure of the beam tends to blow them down the optical axis. Under conditions where the gradient force dominates, a particle can be trapped, in three dimensions, near the focal point.⁷⁹ Reprinted by permission from Macmillan Publishers Ltd: Nature, D. G. Grier, Nature, 2003, 424, 810–816, copyright 2003.

Recently, Laakso and co-workers used an optical trap to measure the displacement generated by single myosin molecules suggesting the use of myosin as a molecular force sensor.⁸² Optical tweezers have been extensively used in in vitrofertilization due to their ability to transport and modify cells precisely.^83,84

The main limitations of optical tweezer based micro/nanomanipulation include: (1) the minimum single object size trapped stably is around 50 nm; (2) precision, distributed, and high speed x-y-z position control of each trap is challenging; and (3) assembled objects should be bonded by chemical or other means to form a mechanically strong and stable material. On the other hand optical manipulation can be particularly advantageous in the handling of biological material in a sterile environment which is often the case in biomedical research.⁸⁵

Manipulation using acoustic forces

Ultrasonic standing wave manipulation is a non-contact mode of particle handling. The non-contact characteristic made this method attractive for use in the manipulation of biostructures since stress induced on the sample is minimal.⁸⁶ Different studies showed that manipulation of biological samples using ultrasound is harmless.^87–89

Ultrasonic waves are generated by using either two opposing sound forces or by a single ultrasonic transducer facing a sound reflector. One commonly used source of ultrasonic waves is piezoceramic elements that are coupled directly into the liquid or via a coupling layer. Free flow acoustophoresis is a separation technique that uses acoustic waves to separate micrometer-size or smaller particles into multiple fractions in a continuous flow mode. These technique have been use to manipulate biological samples such as red blood cells and platelets.⁹⁰ Acoustic trapping of neural stem cells was performed using a microfluidic perfusion microchip without negative effects on the sample.⁹¹

An interesting application of the acoustic separation is the use of acoustic standing wave forces to separate lipid particles from erythrocytes with the possible clinical implication of reducing neurocognitive complications after cardiopulmonary bypass Fig. 8.^89,92 Unfortunately at the moment the acoustophoretic microchips developed can handle just small volumes, a fact that limits their use in clinical applications.

Fig. 8 Human lipids particles separated from human erythrocytes at the trifurcation of 350 μm separation chip with ultrasound turned on. [F. Petersson et al., Lab Chip, 2005, 5, 20–22] Reproduced by permission of the Royal Society of Chemistry.⁹²

Directed self-assembly

Things can be made in a different way and without a guiding hand: that is, by self-assembly (Fig. 9). Self-assembly was originally defined in molecular systems as a process in which molecules or parts of molecules spontaneously form ordered aggregates, usually by non-covalent interactions.⁹³ Self-assembly offers a new approach to the assembly of multicomponent micro- and nanosystems. In nature, molecular self-assembly is a process by which starting from simple building blocks such as proteins or peptides , complex three-dimensional structures with well-defined functions are constructed.^94,95

Fig. 9 Chart illustrating the general scheme of “making things” at size scales ranging from nanometres to kilometres, and the possible niches for application of mesoscale-assembly. M. Boncheva and G. M. Whitesides, “Making Things by Self-Assembly”, MRS Bulletin, 2005, 30(10), 736, Fig. 1. Reproduced by permission of the MRS Bulletin.⁹⁴http://www.mrs.org/bulletin.

Several characteristics of these processes suggest that self-assembly is technologically important for the various microtechnologies and for material science: (1) Self-assembly allows the organization of small and numerous components into ordered structures (both in 2 and 3-D) in a parallel process. (2) Self-assembly provides access to aggregates with electric/electronic or optical functionality. (3) Components fabricated using incompatible processes and materials can be combined in the same self-assembled aggregate. (4) Self-assembly allows one to achieve very high accuracy of registration in positioning small components. (5) Self-assembly offers opportunities to achieve low defect rates and high yields. (6) Self-assembly is a parallel process and can be fast when large numbers of components are involved.⁹⁴

In directed self-assembly, the components and interactions are tailored to form a desired structure. Electrostatic interactions, mechanical vibration, and surface tension can be used to drive the assembly process on different length scales.

The main challenges of directed self-assembly systems are a need for rigorous predictive models (yield, time and accuracy), a higher yield and faster assembly, programmability and control of the self-assembly, multilayer assembly, and not being limited to specific materials geometry.¹⁹

Manipulation using microfluidics

The development of microfluidic structures for the manipulation and analysis of biological samples is a hot topic due to the many advantages this systems offer: low cost, low power and reactant consumption, portability, potential for parallel operation and integration with other devices, and short time analysis amongst many others. Normally these microfluidic channels are integrated with other components that will complete the analysis of the biological sample. The principal function of the microfluidic component is to focus, separate or bring the sample to the complementary component (magnetic, optical, electrical, mechanical, etc.) which will manipulate and/or detect a signal coming from the sample.⁶⁷

The method by which microfluidic devices can manipulate biological samples is based on mechanical principles (hydrodynamic force, gravity, capillary, wetting and adhesion forces) and the geometry of the microfluidic device.⁹⁶ In this way biological components can be separated, captured, redirected, sedimented, mixed, concentrated or diluted. By using microfluidics devices, large populations or individual cells can be manipulated and analyzed without labelling markers or without modification of the cell behaviour.

By constricting a channel, the so called pinched-flow zone, cells can be aligned and later separated without applying any other external force different that the hydrodynamic flows transporting the sample. Yamada and co-workers demonstrated the use of pinched flow fractionation to separate large cells, eliminating the influence of gravity.⁹⁷ An example of the combination of microfluidics with optical components can be found in the work of Goksor and co-workers. They developed a microfluidic device where optical manipulation, imaging and microfluidics were combined to study single cells.⁹⁸ A successful combination of microfluidics together with optical trapping and fluorescence microscopy allowed the direct observation of individual RecA filaments assembling on single DNA molecules. Galletto and co-workers designed a flow cell where three micro channels were used to capture single DNA molecule and to follow the grow of RecA filaments.⁹⁹ Single DNA molecules were isolated and trapped in a microfluidic device combining microfluidics and dielectrophoresis. The transit time and the number of DNA molecules trapped were optimized by changing the microchannels. By optimizing the dimensions of the microfluidic channels the volume of solution was reduced, improving in this way the transit time and reducing the number of DNA molecules used in the analysis, Fig. 10.¹⁰⁰ By adding posts inside a microfluidic channels separation of tumor cells from whole blood was possible reaching a percentage recovery of 65% from 50 to 5000 cells mL⁻¹.¹⁰¹

Fig. 10 Schematic diagram showing the main elements of a microfluidic device using microchannels and dielectrophoresis. M. Kumemura et al., Chem. Phys. Chem., 2007, 8, 1875–1880. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.¹⁰⁰

Arata and co-workers at Tokyo University present different examples of the use of microfluidics in combination with other techniques for the manipulation of DNA and proteins. In their review they present MEMS-based microsystems as powerful tools in manipulating and analyzing biomaterials at the molecular level.¹⁰²

Besides all the advantages that microfluidic devices offer for the manipulation of biological samples, drawbacks such as time consumption in optimizing flow control, difficult integration with other components (e.g. optical components), fabrication reproducibility, poor selectivity and user interface, are issues that needs to be considered when developing microfluidic devices. Additionally the handling of some biological samples can be tricky as in the case of the interaction of blood with the different polymer materials used in microfluidic device fabrication. The different phenomena occurring when blood and different materials interact such as conformational changes of proteins, adhesion of platelets and thrombus formation, introduce additional challenges to the fabrication of polymer microfluidic devices for the manipulation of blood.¹⁰³

Concluding remarks

Feynman vision mentioned in his famous talk:¹⁷¹ “There’s plenty of room at the bottom”, is close to reality today. Micro and nano tools enable researchers to manipulate small objects. Many of these methods and techniques can be used to manipulate and integrate biological samples to build microbiological machines with applications in diagnosis, biosensing, and bioelectronics. Unfortunately in almost all the cases, due to the technical requirements, the sample cannot be kept in its natural environment or it would suffer some damage. The challenge is then to develop new techniques that allow us to manipulate biological entities without changing their structure. In this way the information obtained from them will be more trustworthy and precise. Some steps have been done already as in the case of the ESEM or the integration of microfluidics with DEP or magnetic fields but still some issues remains to be solve. In this article we have tried to give an overview of all the methods available from the literature, listing their advantages and disadvantages. With the amount of research carried out in this field, it is certain that more methods will be developed in the future, so that eventually we can talk about manipulation techniques for biological samples that will interact with the samples without damaging or changing their natural structures.

Acknowledgements

Funding from the European Community (BeNatural/NMP4-CT-2006-033256) is gratefully acknowledged.

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