Nanomanipulation of Biocells
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KeywordsEnvironmental Scanning Electron Microscope Biological Cell Optical Tweezer Cell Position Piezo Actuator
Nanomanipulation for biocells is the usage of precise manipulation techniques to tackle the fundamental and practical problems in biological cells at small scale, including, but not limited to, cell positioning, cell characterization, cell assembly, and so on.
Biological cell is the basic unit of life. This small organizational system maintains a highly complex and hierarchical architecture of interconnected molecular networks. The analysis on biological cell greatly benefits the basic research in life science and also clinical applications. Nanomanipulation enables to control the cell’s position precisely; thereby, it provides the possibility to study the cell’s properties at very small scale. Nowadays, nanomanipulation has been successfully used in cell positioning, cell characterization, cell assembly, and other biological cell analysis fields.
Key Techniques in Nanomanipulation
Wireless manipulators, including dielectrophoresis (DEP), optical tweezers, magnetic twisting cytometry, and others, avoid the usage of electrical wires (Fig. 1b–d). Thus, they are able to manipulate the sample remotely without physical contact, which provides the possibility for the biological cell analysis in a closed environment. Nowadays, these manipulation systems have been widely used for the cell analysis in the microchip, including for cell array, cell characterization, and so on . One potential problem of the wireless manipulation is that they have special requirements on the manipulation environment, such as light transmission solution, magnetic objects, and so on. Another problem is force-producing ability of this kind of system is relative low, i.e., usually less than micro-Newton. Thus, the application area of the wireless manipulators is not as wide as the piezo actuator.
In addition to the above methods, the bio-inspired manipulator also has fast-rising growth these days. Creatures or the actuation part of creatures have been taken as the manipulator directly, such as sperm, bacteria, flagella, protein, DNA, and so on (Fig. 1e) . These bionanomanipulators have many advantages, such as tiny size, simple structure, self-assembly, and biocompatibility. Inspired by the actuation mechanism of bacteria, an artificial flagella is developed (Fig. 1f) . Although the research in this field is still at the preliminary stage, it is thought that the biochemical-inspired nanomanipulator would play more and more important roles in the future.
MEMS device is one type of successfully applicable end effector for the manipulation of cells at small scale. It is usually designed based on the electrostatic mechanism. Controlling by the input voltage, the different parts of the MEMS device can move relatively. Therefore, the MEMS device enables to do the open and close motion if well designed with two probes, such as microgripper . In addition, some unique materials, such as shape memory alloy and thermal expansion polymer, are also used to improve the movement ability of the MEMS device.
AFM cantilever is another type of widely used end effector for cell manipulation. AFM cantilever can be integrated with the nanomanipulation system easily owing to its small size. More important, AFM cantilever can provide force information during the manipulation process based on the deflection of the cantilever beam. Regarding these advantages, AFM cantilever provides a great opportunity for the cell characterization, including the mechanical, electrical, and other properties. To improve the function of AFM cantilever further, various shapes of end effectors are developed based on AFM cantilever. These end effectors not only keep the force measurement ability but also have specific function according to the design. For instance, by modifying the tip of AFM cantilever via focused ion beam (FIB) etching, different functional end effectors are developed to study the properties of single cell, such as the nanopicker for cell-cell adhesion study, dual probe for cell electricity study, microprobe for cell adhesion strength study, and so on [7, 8, 9].
Examples of Applications
In the direct cell positioning, the end effector, such as micropipette and microgripper, are assembled on the nanomanipulator firstly. Driven by the nanomanipulator, the position of the end effector is able to be precisely controlled. Since the end effector is able to handle and release the cell, this integrated system enables the pick-transport-place of a specific biological cell precisely and effectively. The issue for the direct manipulation is that the end effector has to contact with the cell during the manipulation process. Thus, it is difficult to implement this platform in the closed environment, like inside the microfluidic channel.
Indirect cell positioning method is based on the wireless actuation techniques, which can manipulate the sample remotely without physical contact. Optical tweezers are instruments that use a highly focused laser beam to hold and manipulate small objects. In the late 1980s, Arthur and Joseph demonstrated the first application of optical tweezers to the biological sciences, using it to trap an individual tobacco mosaic virus and Escherichia coli bacterium . Today, optical tweezer has been a common scientific instrument in trapping cells to study a variety of biological problems. Because optical tweezers can manipulate the object precisely without physical contact, it is very useful for the cell positioning inside the microchip. The main issue of the optical tweezers is that the generated force by laser is usually limited to nano-Newton, which is not large enough to drive the nonsuspending cells some time. Dielectrophoresis (DEP) is the phenomenon in which a force is exerted on a dielectric particle when it is subjected to a nonuniform electric field. DEP has the advantages of noncontact manipulation, good controllability, and high integration ability with the microchip. Therefore, it is another powerful technique for the cell trapping and sorting, especially for the cell analysis in the microfluidic chip. The drawback of DEP is that the strength of the force depends strongly on the medium and particles’ electrical properties, on the particles’ shape and size, as well as on the frequency of the electric field. Another problem is that it is difficult to make the size of the electrode to nanoscale, which affects the manipulation accuracy. To overcome these drawbacks, a new concept called optoelectronic tweezers (OETs) is proposed . OETs use projected optical images to grab and corral tiny particles with sizes ranging from hundreds of micrometers to tens of nanometers. In this method, light first creates “virtual electrodes” on the substrate. Then, the image in conjunction with an externally applied electrical bias creates the localized DEP traps in the illuminated areas. This method combines the advantages of optical tweezers and electrode-based DEP, which is expected to have a bright future in the cell positioning.
Cell adhesion is a very important research topic in biological field, since many cell activities depend on the attachment of cells to the extracellular matrix or neighboring cells. One traditional nanomanipulation technique for cell adhesion study is micropipette aspiration . In this method, two aspiration pipette end effectors are assembled on two micromanipulators, respectively. The negative pressure from the pipette allows the cell to be grasped on the tip. Driven by the nanomanipulator, the two pipettes pull the cells from opposite direction to separate them. With the increasing of the suction force gradually, the cell would be separated finally. In this process, the cell-cell adhesion strength is able to be calculated based on the suction pressure. AFM system is another powerful nanomanipulation system to study the cell’s adhesion properties, including both the cell-substrate and the cell-cell adhesion [13, 14]. Here, AFM cantilever is not only the end effector to manipulate the cell but also the sensor to measure the adhesion force. One common setup is to use the AFM cantilever to detach the cell on the sample stage, in which the adhesion strength is measured based on the deflection of the AFM cantilever. If one cell is immobilized on a modified AFM cantilever tip, the adhesion cell-cell force can be measured, in which process the specific tip is used to press on and leave from another cell. The adhesion between these two cells can be calculated based on the deflection of the AFM cantilever. In addition, AFM nanomanipulation system also allows the adhesion strength measurement between the cell and the proteins. In this process, the AFM cantilever tip is modified with protein first. Then, this cantilever is used to study the cell-protein adhesion following the same process of cell-cell adhesion measurement. AFM system has the advantages of higher positioning accuracy and higher force resolution. One main issue is that the AFM system cannot scan the sample to get high-resolution image during the adhesion characterization process. Environmental scanning electron microscope (ESEM) is one unique type of SEM. It can not only keep the real-time observation ability of SEM but also allow the nonconductive and aqueous sample observation. Integrating with the nanomanipulation technique, ESEM provides a novel approach for single-cell analysis [1, 15]. AFM cantilever–based end effector is usually used in this characterization system. By modifying the AFM cantilever with nanofabrication technique, various end effectors can be developed for different characterization tasks, such as the microputter and nanopicker for the cell-substrate and cell-cell adhesion measurement respectively [7, 8]. This system allows the real-time imaging at nanometer resolution during the manipulation, which provides more information to benefit the single-cell characterization.
Cell stiffness and elastic characterization is another application example of the nanomanipulation system. Recently, various nanomanipulation methods have been reported to study single cell stiffness, including MEMS tool, optical tweezers, magnetic beads, AFM system, ESEM nanotool, and so on. MEMS tool is an effective end effector for cell characterization owing to its force measurement ability. For example, an integrated manipulator and MEMS probe system is developed to measure the stiffness of the pollen tube cell . This characterization system allows measuring the local region of the cell, but the probe size of the MEMS tool is difficult to be at nanoscale. Optical tweezers enable high positioning accuracy and high force resolution for the stiffness characterization. It has been successfully used to study the mechanics of the human red blood cell . The relative low force output ability (~pN) and light-induced damage are thought as the challenges for optical tweezer manipulation. Magnetic manipulation drive small beads to deform the cell based on the controlling of the magnetic field, which avoids potential light-induced damage as in laser trapping. The local viscoelastic value of the cell can be measured based on the applied magnetic strength on the beads. In this method, the modeling and controlling of the nonhomogeneous magnetic field are considered as the challenge. The high force sensitivity of the AFM system makes it particularly suitable for cell stiffness study . Here, the AFM cantilever is used as the nanoindentor to deform the cell. Then, the stiffness value is calculated based on the cell deformation and the applied force. One potential problem in this method is the pyramid structure of AFM tip, which causes difficulty to the mechanical modeling when the cell has a large deformation. To overcome this drawback, a nanoneedle with a long slim probe is designed based on FIB etching technique . Driven by the nanomanipulator, the nanoneedle allows a large deformation on the cell surface, thereby to provide more accurate mechanical models for the cell.
Nowadays, the characterization of a cell’s electrical property has attracted increasing interest, because it reflects the physiological state of the cell and has wide application in medical field. To study the electrical properties of the cell accurately, a precise nanomanipulation system and a tiny conductive probe are required. An integrated system, consisting of robot-assisted AFM nanomanipulation system and the traditional patch-clamp system, is able to measure the electrical response of the human cell corresponding to external mechanical stimulation . This system expects to benefit the drug test in the future. More than the single probe, an end effector with dual probes is also developed to study the electrical resistance of the cell . Driven by the nanomanipulation system, the dual probes penetrate the cell wall first. Then, the current between the two probes is measured by applying a low voltage. This experiment setup can detect the resistance difference of living and dead cells, which expects to have wide applications in cell biology study.
As the basic unit, a cell is always considered as the “block” of life. How these blocks build life is one of the biggest questions in the world. Therefore, increasing interests have been focused on the assembly of 3D biostructure from single cells.
Generally, a cell requires a relative strong “scaffold” to support its growth in 3D space. Thus, the preparation of 3D scaffold is one key step in cell assembly. 3D printing is a novel technique that combines robotic manipulation and material science together. It is able to print the scaffold or components with single cells to 3D structure directly. With the support of nanomanipulation, 3D printing can also work at small scale. For instance, a scaffold with structure at nanoscale is fabricated based on nanoscribe technique, which is successfully used to study the growth of single cells in 3D space . In addition to 3D printing, other fabrication techniques are also applicable for 3D cell assembly, such as the controllable optical induced method. Because some gels are sensitive to light, the formation of the gel can be controlled by light. On the basis of this principle, 3D gel has been generated successfully with different shape and height by controlling the light illumination area and the exposure time .
Another assembly method is to directly build the blocks with cells to 3D structure, as house building. First, the biological cells are capsulated in series gel blocks by biochemical process. Then, small robots actuated by magnetic fields are used to manipulate these blocks to build complex 3D structures . To improve the manipulation ability, the biocompatible magnetic nanoparticles can also be mixed in the gel, so the position of the gel can be manipulated by magnetic fields more flexibly.
Nanomanipulation technique has greatly benefited the biological cell research from many aspects. However, there are still lots of challenges for the manipulation system. The first one is the actuation technique for the nanomanipulator, which covers power supply, energy transmission, driven approach, and so on. To address the problems in practice, the actuation system for the nanomanipulator should be much smaller, higher output capability, and higher efficiency. Biological creature is the “nanorobot” in nature, which has high working efficiency and exactly compatible for bioapplication. “Learn from nature” will drive new inspiration to the nanomanipulation, which is expected to benefit the biomedical field in the future. Secondly, the data with high spatial and time resolution would help us to understand the cell activities better. Nowadays, people have shown increasing interest to cell research at the subcellular or molecule level. Thus, the nanomanipulation system should be much more accurate and intelligent to meet the above requirement. Last, the commercialization of the nanomanipulation system should be considered. Although many exciting nanomanipulation techniques have been reported, most of them are still in the laboratory. How to bring these techniques to market to benefit our society will be a significant task in the future.
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