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In situ sensing and manipulation of molecules in biological samples using a nanorobotic system
Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 31 – 40 www.nanomedjournal.com
Basic Research
In situ sensing and manipulation of molecules in biological samples using a nanorobotic system Guangyong Li, MS,a Ning Xi, PhD,a,T Donna H. Wang, MDb a
College of Engineering, Department of Electrical and Computer Engineering, Michigan State University, East Lansing, MI 48824 b Department of Medicine, Michigan State University, East Lansing, Michigan Received 5 October 2004; accepted 23 November 2004
Abstract
Background: Atomic force microscopy (AFM) is a powerful and widely used imaging technique that can visualize single molecules both in air and solution. Using the AFM tip as an end-effector, an atomic force microscope can be modified into a nanorobot that can manipulate objects in nanoscale. Methods: By functionalizing the AFM tip with specific antibodies, the nanorobot is able to identify specific types of receptors on cells’ membrane. It is similar to the fluorescent optical microscopy but with higher resolution. By locally updating the AFm image based on interaction force information and objects’ model during nanomanipulation, real-time visual feedback is obtained through the augmented reality interface. Results: The development of the AFM-based nanorobotic system will enable us to simultaneously conduct in situ imaging, sensing, and manipulation at nanometer scale (eg, protein and DNA levels). Conclusions: This new technology opens a promising way to individually study the function of biological system in molecular level. D 2005 Published by Elsevier Inc.
Key words:
Single molecule recognition; AFM; Nanomaniplation; Augmented reality
The daunting challenge that we are facing in the postgenome era is to understand gene and protein functions. Tremendous efforts have now been directed toward the development of gene and protein expression profiling, which allows us to look at multiple factors involved in diseases such as essential hypertension, known as polygenic disease. However, this bglobal approachQ merely gives a snapshot of a sequence of events and may not provide cause-and-effect analysis. Fortunately, the development of atomic force microscopy (AFM) [1] opens a new way to study the functions of the single molecules of genes and proteins individually. The AFM technique was developed initially as an instrument mainly used for surface science research. Research efforts in the past few years have indicated that AFM is also a potentially powerful tool for biochemical and
T Corresponding author. E-mail address: [email protected] (N. Xi). 1549-9634/$ – see front matter D 2005 Published by Elsevier Inc. doi:10.1016/j.nano.2004.11.005
biologic research [2]. This rapid expansion of AFM studies in biology/biotechnology results from the fact that AFM techniques offer several unique advantages. First, they require little sample preparation, with native biomolecules usually being imaged directly. Second, they are less destructive than other techniques (eg, electron microscopy) commonly used in biology. Finally, they can operate in several environments, including air, vacuum, and liquid, and even when the cells are still alive [3]. However, studies of living cells using AFM with high resolution are hampered by cell deformation and tip contamination [4]. Various approaches have been used to obtain high-resolution images of soft biologic materials. At low temperature, cells stiffen and high-resolution imaging becomes feasible [5]. Cells also become stiff after chemical fixation [6]. These circumstances, however, can hardly be called physiologic. Another solution is to use tapping mode AFM (TMAFM) in liquid, which gives a substantial improvement in imaging quality and stability over the standard contact mode [7]. Because of the viscoelastic properties of the plasma membrane, the cell
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Environment Model
Local Scan
Interactive Force
+ Updated AFM Image
Augmented Reality Environment
Haptic
AFM System
Feedback
Command Generator
Fig 1. AFM-based nanorobotic system, which includes an AFM system (nanorobot) and the augmented reality environment (visual haptic interface and command generator).
may behave like a bhardQ material when responding to externally applied, high-frequency vibration; effectively, it is less susceptible to deformation [4]. Recent progress in the spatial resolution of AFM technology has made topographic imaging of a single protein routine work [8,9]. However, it is still impossible to recognize specific proteins such as receptors only from topographic information. Because the interaction between ligands and receptors is highly specific and possesses a high degree of spatial and orientational specificity, the technique to functionalize an AFM tip with certain molecules has opened a promising way to recognize single specific molecules. It has been proven that single receptors can be recognized by an AFM tip functionalized with an antibody through a force mapping technique [10 -12]. However, all such results are obtained by imaging wellprepared samples on substrate surfaces instead of under physiologic conditions. In practice, it is still very difficult to obtain clear imaging of living cells on a molecular level via AFM. In addition to the capability of AFM to characterize surfaces in nanometer scale, it has been demonstrated recently that AFM can be used as a nanorobot to modify surfaces and manipulate nanosized structures by using the AFM tip as an end effector [13 -16]. The main problem with using the AFM as a manipulator is the lack of realtime visual feedback during manipulation. Fortunately, this problem can be solved by a recently developed AFM-based nanorobotic system with an augmented reality interface [17]. The system aims to provide the operator with both real-time visual display and real-time force feedback. The real-time visual display is a dynamic AFM image of the operating environment, which is locally updated based on real-time force information, system
models, and local scanning information. However, most studies on nanomanipulation using AFM were carried out in ambient conditions, and few of them under a liquid. In this article, we examine the angiotensin II type 1 receptor (AT1) on living neuron cells using the end effector functionalized with the AT1 antibody. Imaging and manipulation of living neuron cells under liquid is discussed using the AFM-based nanorobotic system, and imaging and manipulation of single DNA molecules are also discussed. Living cell images are obtained in their physiologic environments using TMAFM. Under the assistance of the augmented reality system, manipulations of DNA molecules and living neuron cells are performed at the nanoscale level. The novel sensor-based intelligent processing and control schemes incorporated within the AFM-based robotic system allow us to achieve reliable and precise manipulations to enable direct investigation of the functions of single molecules or proteins and their signaling pathways in specific cells or tissues both in vivo and in vitro.
Material and methods AFM-based nanorobotic system The AFM-based nanorobotic system includes 2 subsystems: the AFM system and augmented reality environment. The AFM system (bottom right, Figure 1) is the main part of the nanorobotic system designed for imaging and manipulation. The augmented reality environment (bottom middle, Figure 1) provides the operator a real-time interactive environment to control the tip motion through a haptic joystick, while viewing the real-time AFM image and feeling the real-time force feedback during manipulation. The real-time visual display is a dynamic AFM image of the
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Fig 2. AFM images of DNA samples. A, Image of DNA sample in scanning range of 5 Am. Most DNA molecules are longer than 5 Am. B, Image of DNA sample in scanning range of 2 Am.
operating environment, which is locally updated, based on the real-time force and the local scanning information as well as the object’s behavior models. The AFM system called Bioscope (Digital Instruments Inc) is equipped with a scanner with a maximum XY scan range of 90 Am 90 Am and a Z range of 5 Am. Peripheral devices include an optical microscope, a charge-coupled device (CCD) camera, and a signal access module that can access most real-time signals inside the AFM system. The inverted optical microscope and the CCD camera help the operator to locate the tip, adjust the laser, and search for interesting areas on the substrate. The augmented reality environment is implemented in another computer equipped with a haptic device (Phantom; SensAble Technologies, Woburn, Mass). With use of a functionalized AFM tip as the end effector, the AFM-based nanorobotic system can identify single biomolecules directly from a living cell’s membrane surface and possibly manipulate these biologic macromolecules in their physiologic environment. End-effector functionalization with antibody Functionalization of AFM tips by chemically and biologically coating them with molecules has opened a new research area for studying specific interactions on a molecular level, such as the binding force of biotin–avidin pairs [18,19] and antigen–antibody pairs [20,21]. Chemical coating of probes is mainly done by silanization or by functionalized thiols and is often the first step before biological functionalization. Many protocols can be performed to attach proteins to a tip. There are 2 main ways to functionalize the AFM tips with antibodies. One method is to directly coat the antibody on a silanized tip. Another method is to tether the antibody on a tip by a linker. The
direct coating method is simple, and results in high lateral resolution. The tethering method needs a much more complicated protocol, but it results in better chance of antigen recognition because the interaction between the antibody and antigen is highly specific and processes a high degree of spatial and orientation specificity. The drawback of the tethering method is that lateral resolution is decreased. Detailed processes of functionalization are presented in the following subsections. Direct coating method The direct coating method is based on silanizing a solid surface with 3-aminopropylmethyl-diethoxysilane (APrMDEOS) (Sigma-Aldrich, St Louis, Mo), which protonates at neutral pH. The silane group in APrMDEOS is highly reactive, and silanizes the surface by forming covalent bonds with surface atoms. Briefly, the silicon nitride tips are treated with 10% nitric acid solution and left in a silicone bath for 20 minutes at 808C. This causes the formation of surface hydroxyl groups on the SiN tips. The tips are then thoroughly rinsed with distilled water, placed in 2% APrMDEOS solution in toluene, and kept in a desiccator purged with argon gas for 5 hours. This treatment provides reactive primary amine groups on the nitride surface. The tips are washed thoroughly with PBS and placed in a solution of 2 Ag/mL anti-AT1 IgG for 10 minutes. The antibody-conjugated tips are then washed thoroughly with PBS and distilled water to remove loosely attached antibodies. These tips are used immediately without being dried. Tethering method The functionalization of the AFM tip with antibody using tethering method involves many more steps than the direct coating method. A spacer to covalently bind the proteins is
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Fig 3. Left, AFM head operated under liquid. Right, Optical image of living neuron cells with low magnification (the AFM tip is adjusted to the top of the cell surface).
usually required to orient the protein to expose specific site(s) of the proteins. Polyethyleneglycol (PEG) is a common spacer. A terminal thiol group can first be attached to PEG, and this thiol group can bind to a gold-coated silicon nitride tip. An amine group at the other end of the PEG molecule attaches proteins (eg, antibodies) via a covalent bond [20]. Biologic coating has been mostly used for measuring the binding force between a receptor and a ligand [22,23], including antigen-antibody pairs [21] as well as mapping the distribution of binding partners on samples [10 -12]. The protocol of functionalization using the tethering method can be found in Hinterdofer et al [20]. Briefly, it can be completed by the following steps: (1) modify the AT1 antibody with N-succinimidyl 3-acetylthiopropionate (SATP, Sigma-Aldrich) by adding a 10-fold molar excess of SATP/DMSO solution to the desalted antibody solution and then using a PD-10 column (Amersham Biosciences, Piscataway, NJ) to collect the modified antibody; (2) modify AFM tips with aminopropyltriethoxysilane (APTES, SigmaAldrich) by evaporating APTES and N,N-diisopropylethyl-
amine (99%, distilled, Sigma-Aldrich) under argon gas to let the gas deposit on the cleaned cantilevers; (3) tether the cross-linkers or spacer (NHS-maleimide, Nektar, Huntsville, Ala) on tips by mixing the cross-linkers and 5 AL of thiethylamine (Alfa Aesar, Ward Hill, Mass) in 1 mL of CHCl3 and then placing the NH2-modified tips into this solution for 2 to 3 hours; and (4) link the SATP-labeled antibody to tips by incubating the tips in 50 AL of SATPlabeled antibody, 25 AL of NH2OH reagent (500 mmol/L NH2OH d HCl/25 mmol/L EDTA, pH 7.5), and 50 AL of buffer A for 1 hour. Finally, the functionalized tips are stored in PBS buffer at 48C for future use. Cell sample preparation The living cell samples are neural cells growing on the glass coverslips in diameter of 15 mm. The cells are originating in the dorsal root ganglia (DRG) tissue of male Wistar rats (body weight, 125 to 200 g). The DRGs from the cervical, thoracic, lumbar, and sacral levels were removed aseptically and collected in F12 medium (Gibco/BRL,
Fig 4. Low-magnification image of the living cells using the TMAFM with scanning range of 25 Am. Left, Height image. Right, Phase image.
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Digital Instruments NanoScope Scan size 1.000 µm Scan rate 0.2913 Hz Number of samples 256 Image Data Phase Data scale 20.00 ° Engage X Pos -19783.4 µm Engage Y Pos -42151.3 µm
view angle light angle
µm 0.8 0.6 0.4 0.2
x 0.200 µm/div z 20.000 °/div
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Fig 5. Three-dimensional view of high-magnification imaging of the living cell using TMAFM with scanning range of 1 Am.
Gaithersburg, Md). The trimmed DRGs were digested in 0.25% collagenase (Boerhinger Mannheim, Indianapolis, Ind) in F12 medium at 378C for 90 minutes. After a 15-minute incubation in PBS containing 0.25% trypsin (Gibco/BRL), the tissues were triturated with a pipette in F12 medium containing DNAse (Sigma-Aldrich, 80 Ag/mL), trypsin inhibitor (Sigma-Aldrich, 100 Ag/mL) and 10% heatinactivated horse serum (Hyclone, Logan, Utah). Cells were then seeded in a 12-well culture plate with polyornithinecoated glass coverslides inside. The cells were cultured in a humid incubator at 378C with 5% carbon dioxide and 9% air.
The cells are ready for AFM scanning after 7 to 10 days of culture. DNA sample preparation DNA plays a key role in biology as a carrier of genetic information in all living species. The Watson-Crick doublehelix structure for DNA has been known for almost 50 years. During the last half-century, the majority of research in DNA has been devoted to its biologic properties, especially its role in genetic inheritance, disease, aging, RNA synthesis and mutation. The heavy-water adsorption of DNA molecules,
Fig 6. The AFM image of living neuron cells with scanning rang of 90 Am. Left, Topographic image. Right, Phase image.
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Fig 7. Left, Augmented reality environment in which the real-time visual and haptic feedback are provided. Right, Real-time image displayed in the augmented reality interface.
because of the negatively charged backbone of the DNA helix structure, causes a strong adhesive force between the DNA molecules and a hydrophilic surface, which destroy the double-helix structure of the DNA molecules. By measuring the height of a single DNA molecule, it has been found that there is very large compression deformation of the deposited DNA on the most commonly used substrates such as the mica and silicon oxide surface [24]. According to Kasumov et al. [25], the thickness of DNA molecules on a silicon substrate treated by pentylamine is about 2.4 nm while the thickness is about 1.1 nm on a clean substrate. Therefore, the E-DNA molecules (Sigma-Aldrich) are deposited on a hydrophobic polycarbonate surface to avoid the compression deformation due to adhesive force. AFM images of DNA samples are shown in Figure 2. Results and discussion Imaging living cells The glass coverslip with a monolayer of DRG cells grown on the surface was put into a petri dish containing F12 medium. A single cell was located using the optical
microscope and then moved underneath the cantilever tip, as shown in Figure 3, by adjustment of the AFM stage. The image of the living cells was obtained using the tapping mode AFM (Figure 4). An image with higher magnification was obtained by zooming in the top of the cell membrane around the nucleus (Figure 5). Manipulation of living cells After the AFM images of the living cells are obtained, manipulation can be performed under the assistance of the augmented reality system. The tip can be injected into the cell or cut the cell membrane at certain locations. Figure 6 shows neuron cells imaged in TMAFM. Figure 7 shows the real-time image displayed in augmented reality environments during manipulation. The big circle in Figure 8 is the first try at cutting a large neuron cell axon. The small circle in Figure 8 is the second attempt to cut a small neuron cell branch. It can be seen that the final results of the cutting operation obtained from the AFM image are consistent with those displayed in the augmented reality environment. Manipulation of live cells under liquid can also be performed at a very small-scale range.
Fig 8. Final result of the cutting operation obtained from AFM image with scanning range of 90 Am. Left, Height image. Right, Phase image.
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Digital Instruments NanoScope Scan size 500.0 nm Scan rate 0.2963 Hz Number of samples 256 Image Data Phase Data scale 20.00 ° Engage X Pos -19783.4 µm Engage Y Pos -42151.3 µm
view angle light angle
nm
400 300 200 x 100.000 nm/div z 20.000 °/div
100
0°
neuron_9_10_2004.008_1
Fig 9. Three-dimensional view of a phase contrast imaging of the living-cell membrane surface using TMAFM with scanning range of 500 nm. The image is obtained using a functionalized tip with AT1 antibody and then passing through a bandpass filter. The AT1 receptors are clearly identified as shown inside the circles.
Single molecules recognition
graphic information is difficult. Fortunately, the new technique, tapping-mode phase imaging, provides a promising solution. It can differentiate between areas with various properties regardless of their topographic nature [26,27]. The
It is well known that recognizing specific proteins such as receptors on the cell membrane surface only from topo-
Digital Instruments NanoScope Scan size 500.0 nm Scan rate 0.2913 Hz Number of samples 256 Image Data Phase Data scale 10.00 ° Engage X Pos -19783.4 µm Engage Y Pos -42151.3 µm
view angle light angle
nm
400 300 200 100
x 100.000 nm/div z 10.000 °/div
0°
neuron_9_10_2004.021
Fig 10. Three-dimensional view of phase contrast imaging of the living cell membrane using TMAFM with scanning range of 500 nm. The image is obtained using a regular tip and then passing through the same bandpass filter as that used in Figure 9. However, there are no receptors can be identified in the image.
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Fig 11. A, AFM image of DNA ropes in its original shape. B, DNA ropes are cut by the AFM tip. The pushing force can be controlled to cut the DNA rope or only deform the DNA rope. The big scratches on the surface indicate strong pushing force applied, and small scratches imply a small pushing force. Arrows indicate the pushing directions.
phase angle is defined as the phase lag of the cantilever oscillation relative to the signal sent to the piezo driving the cantilever. Theoretic simulations and experiments of the cantilever dynamics in air have shown that phase contrast arises from differences in the energy dissipation between the tip and the sample. The phase shift is related analytically to the energy dissipated in the tip sample interaction, according to the following equation [26,27]: x A QED sinW ¼ þ x0 A0 pkAA0 where W is the phase angle, x/x0 is the working frequency/ resonance frequency, A/A 0 is the set-point amplitude/free amplitude, Q is the quality factor, E D is the energy dissipation, and k is the cantilever spring constant. The phase shift due to the tip-sample interaction, which involves energy dissipation, is the displacement of the noncontact solution to higher phase shifts and the intermittent contact solution to lower phase shift values. The more dissipative y C b A a
O
B
t x
Fig 12. Modeling deformation of DNA molecules and bundles under pushing by the AFM tip. Solid curve, DNA molecule (or DNA bundle) in its original status; dashed curve, new status of the DNA molecule. The DNA molecule (bundle) is pushed by the AFM tip from the start point O to the end point C. The DNA molecule (bundle) is only deformed within an affecting region from point A to B, which is bounded by a circle with radius of a; t is the thickness of the DNA molecule (bundle).
features will appear lighter in the noncontact regime, whereas they will appear darker in the intermittent contact regime [28]. When scanning the cell membrane surface using a tip functionalized with AT1 antibody, the tip-sample interaction force will increase when the tip closes to the AT1 receptor, resulting in a significant change of the phase shift. Because the topographic information is also convoluted to the phase contrast image but at a low frequency, a bandpass filter can be used to remove the low-frequency topographic information and high-frequency noise. After filtering the phase contrast image, only receptor images will be left on the surface. Figure 9 shows a phase contrast image of a neuron membrane obtained using the functionalized tip and then processed with a band-pass filter. The AT1 receptors are clearly indicated on the image. By using the optical scope equipped with the AFM system, the same cell can be scanned using a regular tip without any functionalization. By using the same band-pass filter to process the phase contrast image obtained with the regular tip, no receptor can be identified on the surface Figure 10. These experimental results show that single biomolecules like the receptors on the cell membrane surface can be recognized using the biologically functionalized tip. Manipulation of single DNA molecules using augmented reality system Because multishaped DNA molecules may have diverse properties, artificially modifying the molecules is necessary in the study of DNA properties. Kinks and deformation of DNA molecules can be created artificially using the AFMbased nanomanipulation system by controlling the pushing force between the tip and sample surface. The DNA molecules or DNA bundles can be either broken or deformed (Figure 11). A large pushing force in the normal direction usually breaks the DNA molecule, and a small pushing
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A
B
3.00 µm
0 Data type Z range
Height 50.00 nm
C
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itself, and f can be a fourth-order polynomial or a hyperbolic cosine function. In order to display the DNA deformation in real time, a local coordinate system is defined such that y-axis is along the pushing direction. The original shape of DNA inside the effect circle can be removed and then the new shape of DNA can be displayed with respect to the local coordinate system. By using the model developed in this section, manipulation of DNA molecules can be displayed in real time in the augmented reality environment. An example of DNA manipulation is shown in Figure 13, in which Figure 13, A, shows the DNA molecules in their original shapes, Figure 13, B, shows the manipulation of DNA molecules displayed in the augmented reality environment, and Figure 13, C, shows an AFM image after manipulation. It can be seen that several kinks have been created by slightly pushing the DNA molecules or bundles, and the kinks created in the augmented reality environment are relatively identical to the real results. Conclusions
0 Data type Z range
3.00 µm Height 50.00 nm
Fig 13. Pushing DNA on a polycarbonate surface (scanning range of 3 Am). A, Image of DNA before pushing. B, Real-time display on the augmented reality during pushing. C, A new scanning image after several pushing operations.
force may only deform the DNA molecule without breaking it. In Figure 11, B, the big scratches on the surface indicate a substantial pushing force applied on the AFM tip, and small scratches imply a small pushing force. The DNA bundle was broken when a big pushing force was applied but only deformed when a small pushing force was applied. To display in real time the deformation of DNA molecules or bundles in the augmented reality environment of the nanorobotic manipulation system, the deformation model of DNA molecules and bundles has to be identified. Although some forces dominant at the nanoscale level can be theoretically calculated, it is not feasible to compute them because some required model parameters are not available. Therefore, instead of using mathematic models for the force deformation of DNA molecules, an empirical method is adopted in this research. As shown in Figure 12, the DNA molecule (bundle) is pushed by the AFM tip from the start point O to the end point C. The DNA molecule (bundle) is only deformed within an affecting region from point A to B, which is bounded by a circle with radius of a. The radius is determined by the following empirical function, a = f(b,t), where b is the pushing distance before DNA broken, t is the thickness of DNA
The technique of using a functionalized tip to measure the interaction force between ligands and receptors by AFM has been discussed for more than a decade. Single-molecule recognition on well-prepared samples using a functionalized tip is also achieved in recent research. However, none of these techniques make possible the recognition of single molecules directly from a cell membrane surface. In this article, single AT1 receptor recognition directly from the cell membrane surface is achieved by use of an AFM tip biologically functionalized with the AT1 antibody. By passing the phase images into a bandpass filter, single receptor molecules are clearly identified in the processed image. Using this technique, further study of the trafficking behavior of the AT1 receptor is possible. In this article, an AFM-based nanorobotic system is also introduced, which can be operated both in the air and liquid conditions. Because the single receptor molecules can be identified in their physiologic condition, manipulation of single molecules on a living cell membrane surface also becomes possible. Our future research will show the efficiency of the proposed AFM-based nanorobotic system on manipulating single micromolecules such as receptor proteins in physiologic conditions. The controlled manipulation of selected molecules by AFM combined with high-resolution images will become a powerful approach to gaining information on the molecular interactions occurring within a biomolecular assembly or between biomolecular assemblies, thereby revealing the functionality of an individual biomolecule. We anticipate that the research that seeks to understand and exploit the interaction forces between nanoprobing mechanisms will provide a leap forward for biomedical research, whose progress is limited by the cumbersome and static multistep methods currently available.
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Basic Research
In situ sensing and manipulation of molecules in biological samples using a nanorobotic system Guangyong Li, MS,a Ning Xi, PhD,a,T Donna H. Wang, MDb a
College of Engineering, Department of Electrical and Computer Engineering, Michigan State University, East Lansing, MI 48824 b Department of Medicine, Michigan State University, East Lansing, Michigan Received 5 October 2004; accepted 23 November 2004
Abstract
Background: Atomic force microscopy (AFM) is a powerful and widely used imaging technique that can visualize single molecules both in air and solution. Using the AFM tip as an end-effector, an atomic force microscope can be modified into a nanorobot that can manipulate objects in nanoscale. Methods: By functionalizing the AFM tip with specific antibodies, the nanorobot is able to identify specific types of receptors on cells’ membrane. It is similar to the fluorescent optical microscopy but with higher resolution. By locally updating the AFm image based on interaction force information and objects’ model during nanomanipulation, real-time visual feedback is obtained through the augmented reality interface. Results: The development of the AFM-based nanorobotic system will enable us to simultaneously conduct in situ imaging, sensing, and manipulation at nanometer scale (eg, protein and DNA levels). Conclusions: This new technology opens a promising way to individually study the function of biological system in molecular level. D 2005 Published by Elsevier Inc.
Key words:
Single molecule recognition; AFM; Nanomaniplation; Augmented reality
The daunting challenge that we are facing in the postgenome era is to understand gene and protein functions. Tremendous efforts have now been directed toward the development of gene and protein expression profiling, which allows us to look at multiple factors involved in diseases such as essential hypertension, known as polygenic disease. However, this bglobal approachQ merely gives a snapshot of a sequence of events and may not provide cause-and-effect analysis. Fortunately, the development of atomic force microscopy (AFM) [1] opens a new way to study the functions of the single molecules of genes and proteins individually. The AFM technique was developed initially as an instrument mainly used for surface science research. Research efforts in the past few years have indicated that AFM is also a potentially powerful tool for biochemical and
T Corresponding author. E-mail address: [email protected] (N. Xi). 1549-9634/$ – see front matter D 2005 Published by Elsevier Inc. doi:10.1016/j.nano.2004.11.005
biologic research [2]. This rapid expansion of AFM studies in biology/biotechnology results from the fact that AFM techniques offer several unique advantages. First, they require little sample preparation, with native biomolecules usually being imaged directly. Second, they are less destructive than other techniques (eg, electron microscopy) commonly used in biology. Finally, they can operate in several environments, including air, vacuum, and liquid, and even when the cells are still alive [3]. However, studies of living cells using AFM with high resolution are hampered by cell deformation and tip contamination [4]. Various approaches have been used to obtain high-resolution images of soft biologic materials. At low temperature, cells stiffen and high-resolution imaging becomes feasible [5]. Cells also become stiff after chemical fixation [6]. These circumstances, however, can hardly be called physiologic. Another solution is to use tapping mode AFM (TMAFM) in liquid, which gives a substantial improvement in imaging quality and stability over the standard contact mode [7]. Because of the viscoelastic properties of the plasma membrane, the cell
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Environment Model
Local Scan
Interactive Force
+ Updated AFM Image
Augmented Reality Environment
Haptic
AFM System
Feedback
Command Generator
Fig 1. AFM-based nanorobotic system, which includes an AFM system (nanorobot) and the augmented reality environment (visual haptic interface and command generator).
may behave like a bhardQ material when responding to externally applied, high-frequency vibration; effectively, it is less susceptible to deformation [4]. Recent progress in the spatial resolution of AFM technology has made topographic imaging of a single protein routine work [8,9]. However, it is still impossible to recognize specific proteins such as receptors only from topographic information. Because the interaction between ligands and receptors is highly specific and possesses a high degree of spatial and orientational specificity, the technique to functionalize an AFM tip with certain molecules has opened a promising way to recognize single specific molecules. It has been proven that single receptors can be recognized by an AFM tip functionalized with an antibody through a force mapping technique [10 -12]. However, all such results are obtained by imaging wellprepared samples on substrate surfaces instead of under physiologic conditions. In practice, it is still very difficult to obtain clear imaging of living cells on a molecular level via AFM. In addition to the capability of AFM to characterize surfaces in nanometer scale, it has been demonstrated recently that AFM can be used as a nanorobot to modify surfaces and manipulate nanosized structures by using the AFM tip as an end effector [13 -16]. The main problem with using the AFM as a manipulator is the lack of realtime visual feedback during manipulation. Fortunately, this problem can be solved by a recently developed AFM-based nanorobotic system with an augmented reality interface [17]. The system aims to provide the operator with both real-time visual display and real-time force feedback. The real-time visual display is a dynamic AFM image of the operating environment, which is locally updated based on real-time force information, system
models, and local scanning information. However, most studies on nanomanipulation using AFM were carried out in ambient conditions, and few of them under a liquid. In this article, we examine the angiotensin II type 1 receptor (AT1) on living neuron cells using the end effector functionalized with the AT1 antibody. Imaging and manipulation of living neuron cells under liquid is discussed using the AFM-based nanorobotic system, and imaging and manipulation of single DNA molecules are also discussed. Living cell images are obtained in their physiologic environments using TMAFM. Under the assistance of the augmented reality system, manipulations of DNA molecules and living neuron cells are performed at the nanoscale level. The novel sensor-based intelligent processing and control schemes incorporated within the AFM-based robotic system allow us to achieve reliable and precise manipulations to enable direct investigation of the functions of single molecules or proteins and their signaling pathways in specific cells or tissues both in vivo and in vitro.
Material and methods AFM-based nanorobotic system The AFM-based nanorobotic system includes 2 subsystems: the AFM system and augmented reality environment. The AFM system (bottom right, Figure 1) is the main part of the nanorobotic system designed for imaging and manipulation. The augmented reality environment (bottom middle, Figure 1) provides the operator a real-time interactive environment to control the tip motion through a haptic joystick, while viewing the real-time AFM image and feeling the real-time force feedback during manipulation. The real-time visual display is a dynamic AFM image of the
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Fig 2. AFM images of DNA samples. A, Image of DNA sample in scanning range of 5 Am. Most DNA molecules are longer than 5 Am. B, Image of DNA sample in scanning range of 2 Am.
operating environment, which is locally updated, based on the real-time force and the local scanning information as well as the object’s behavior models. The AFM system called Bioscope (Digital Instruments Inc) is equipped with a scanner with a maximum XY scan range of 90 Am 90 Am and a Z range of 5 Am. Peripheral devices include an optical microscope, a charge-coupled device (CCD) camera, and a signal access module that can access most real-time signals inside the AFM system. The inverted optical microscope and the CCD camera help the operator to locate the tip, adjust the laser, and search for interesting areas on the substrate. The augmented reality environment is implemented in another computer equipped with a haptic device (Phantom; SensAble Technologies, Woburn, Mass). With use of a functionalized AFM tip as the end effector, the AFM-based nanorobotic system can identify single biomolecules directly from a living cell’s membrane surface and possibly manipulate these biologic macromolecules in their physiologic environment. End-effector functionalization with antibody Functionalization of AFM tips by chemically and biologically coating them with molecules has opened a new research area for studying specific interactions on a molecular level, such as the binding force of biotin–avidin pairs [18,19] and antigen–antibody pairs [20,21]. Chemical coating of probes is mainly done by silanization or by functionalized thiols and is often the first step before biological functionalization. Many protocols can be performed to attach proteins to a tip. There are 2 main ways to functionalize the AFM tips with antibodies. One method is to directly coat the antibody on a silanized tip. Another method is to tether the antibody on a tip by a linker. The
direct coating method is simple, and results in high lateral resolution. The tethering method needs a much more complicated protocol, but it results in better chance of antigen recognition because the interaction between the antibody and antigen is highly specific and processes a high degree of spatial and orientation specificity. The drawback of the tethering method is that lateral resolution is decreased. Detailed processes of functionalization are presented in the following subsections. Direct coating method The direct coating method is based on silanizing a solid surface with 3-aminopropylmethyl-diethoxysilane (APrMDEOS) (Sigma-Aldrich, St Louis, Mo), which protonates at neutral pH. The silane group in APrMDEOS is highly reactive, and silanizes the surface by forming covalent bonds with surface atoms. Briefly, the silicon nitride tips are treated with 10% nitric acid solution and left in a silicone bath for 20 minutes at 808C. This causes the formation of surface hydroxyl groups on the SiN tips. The tips are then thoroughly rinsed with distilled water, placed in 2% APrMDEOS solution in toluene, and kept in a desiccator purged with argon gas for 5 hours. This treatment provides reactive primary amine groups on the nitride surface. The tips are washed thoroughly with PBS and placed in a solution of 2 Ag/mL anti-AT1 IgG for 10 minutes. The antibody-conjugated tips are then washed thoroughly with PBS and distilled water to remove loosely attached antibodies. These tips are used immediately without being dried. Tethering method The functionalization of the AFM tip with antibody using tethering method involves many more steps than the direct coating method. A spacer to covalently bind the proteins is
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Fig 3. Left, AFM head operated under liquid. Right, Optical image of living neuron cells with low magnification (the AFM tip is adjusted to the top of the cell surface).
usually required to orient the protein to expose specific site(s) of the proteins. Polyethyleneglycol (PEG) is a common spacer. A terminal thiol group can first be attached to PEG, and this thiol group can bind to a gold-coated silicon nitride tip. An amine group at the other end of the PEG molecule attaches proteins (eg, antibodies) via a covalent bond [20]. Biologic coating has been mostly used for measuring the binding force between a receptor and a ligand [22,23], including antigen-antibody pairs [21] as well as mapping the distribution of binding partners on samples [10 -12]. The protocol of functionalization using the tethering method can be found in Hinterdofer et al [20]. Briefly, it can be completed by the following steps: (1) modify the AT1 antibody with N-succinimidyl 3-acetylthiopropionate (SATP, Sigma-Aldrich) by adding a 10-fold molar excess of SATP/DMSO solution to the desalted antibody solution and then using a PD-10 column (Amersham Biosciences, Piscataway, NJ) to collect the modified antibody; (2) modify AFM tips with aminopropyltriethoxysilane (APTES, SigmaAldrich) by evaporating APTES and N,N-diisopropylethyl-
amine (99%, distilled, Sigma-Aldrich) under argon gas to let the gas deposit on the cleaned cantilevers; (3) tether the cross-linkers or spacer (NHS-maleimide, Nektar, Huntsville, Ala) on tips by mixing the cross-linkers and 5 AL of thiethylamine (Alfa Aesar, Ward Hill, Mass) in 1 mL of CHCl3 and then placing the NH2-modified tips into this solution for 2 to 3 hours; and (4) link the SATP-labeled antibody to tips by incubating the tips in 50 AL of SATPlabeled antibody, 25 AL of NH2OH reagent (500 mmol/L NH2OH d HCl/25 mmol/L EDTA, pH 7.5), and 50 AL of buffer A for 1 hour. Finally, the functionalized tips are stored in PBS buffer at 48C for future use. Cell sample preparation The living cell samples are neural cells growing on the glass coverslips in diameter of 15 mm. The cells are originating in the dorsal root ganglia (DRG) tissue of male Wistar rats (body weight, 125 to 200 g). The DRGs from the cervical, thoracic, lumbar, and sacral levels were removed aseptically and collected in F12 medium (Gibco/BRL,
Fig 4. Low-magnification image of the living cells using the TMAFM with scanning range of 25 Am. Left, Height image. Right, Phase image.
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Digital Instruments NanoScope Scan size 1.000 µm Scan rate 0.2913 Hz Number of samples 256 Image Data Phase Data scale 20.00 ° Engage X Pos -19783.4 µm Engage Y Pos -42151.3 µm
view angle light angle
µm 0.8 0.6 0.4 0.2
x 0.200 µm/div z 20.000 °/div
0°
neuron_9_10_2004.020
Fig 5. Three-dimensional view of high-magnification imaging of the living cell using TMAFM with scanning range of 1 Am.
Gaithersburg, Md). The trimmed DRGs were digested in 0.25% collagenase (Boerhinger Mannheim, Indianapolis, Ind) in F12 medium at 378C for 90 minutes. After a 15-minute incubation in PBS containing 0.25% trypsin (Gibco/BRL), the tissues were triturated with a pipette in F12 medium containing DNAse (Sigma-Aldrich, 80 Ag/mL), trypsin inhibitor (Sigma-Aldrich, 100 Ag/mL) and 10% heatinactivated horse serum (Hyclone, Logan, Utah). Cells were then seeded in a 12-well culture plate with polyornithinecoated glass coverslides inside. The cells were cultured in a humid incubator at 378C with 5% carbon dioxide and 9% air.
The cells are ready for AFM scanning after 7 to 10 days of culture. DNA sample preparation DNA plays a key role in biology as a carrier of genetic information in all living species. The Watson-Crick doublehelix structure for DNA has been known for almost 50 years. During the last half-century, the majority of research in DNA has been devoted to its biologic properties, especially its role in genetic inheritance, disease, aging, RNA synthesis and mutation. The heavy-water adsorption of DNA molecules,
Fig 6. The AFM image of living neuron cells with scanning rang of 90 Am. Left, Topographic image. Right, Phase image.
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Fig 7. Left, Augmented reality environment in which the real-time visual and haptic feedback are provided. Right, Real-time image displayed in the augmented reality interface.
because of the negatively charged backbone of the DNA helix structure, causes a strong adhesive force between the DNA molecules and a hydrophilic surface, which destroy the double-helix structure of the DNA molecules. By measuring the height of a single DNA molecule, it has been found that there is very large compression deformation of the deposited DNA on the most commonly used substrates such as the mica and silicon oxide surface [24]. According to Kasumov et al. [25], the thickness of DNA molecules on a silicon substrate treated by pentylamine is about 2.4 nm while the thickness is about 1.1 nm on a clean substrate. Therefore, the E-DNA molecules (Sigma-Aldrich) are deposited on a hydrophobic polycarbonate surface to avoid the compression deformation due to adhesive force. AFM images of DNA samples are shown in Figure 2. Results and discussion Imaging living cells The glass coverslip with a monolayer of DRG cells grown on the surface was put into a petri dish containing F12 medium. A single cell was located using the optical
microscope and then moved underneath the cantilever tip, as shown in Figure 3, by adjustment of the AFM stage. The image of the living cells was obtained using the tapping mode AFM (Figure 4). An image with higher magnification was obtained by zooming in the top of the cell membrane around the nucleus (Figure 5). Manipulation of living cells After the AFM images of the living cells are obtained, manipulation can be performed under the assistance of the augmented reality system. The tip can be injected into the cell or cut the cell membrane at certain locations. Figure 6 shows neuron cells imaged in TMAFM. Figure 7 shows the real-time image displayed in augmented reality environments during manipulation. The big circle in Figure 8 is the first try at cutting a large neuron cell axon. The small circle in Figure 8 is the second attempt to cut a small neuron cell branch. It can be seen that the final results of the cutting operation obtained from the AFM image are consistent with those displayed in the augmented reality environment. Manipulation of live cells under liquid can also be performed at a very small-scale range.
Fig 8. Final result of the cutting operation obtained from AFM image with scanning range of 90 Am. Left, Height image. Right, Phase image.
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Digital Instruments NanoScope Scan size 500.0 nm Scan rate 0.2963 Hz Number of samples 256 Image Data Phase Data scale 20.00 ° Engage X Pos -19783.4 µm Engage Y Pos -42151.3 µm
view angle light angle
nm
400 300 200 x 100.000 nm/div z 20.000 °/div
100
0°
neuron_9_10_2004.008_1
Fig 9. Three-dimensional view of a phase contrast imaging of the living-cell membrane surface using TMAFM with scanning range of 500 nm. The image is obtained using a functionalized tip with AT1 antibody and then passing through a bandpass filter. The AT1 receptors are clearly identified as shown inside the circles.
Single molecules recognition
graphic information is difficult. Fortunately, the new technique, tapping-mode phase imaging, provides a promising solution. It can differentiate between areas with various properties regardless of their topographic nature [26,27]. The
It is well known that recognizing specific proteins such as receptors on the cell membrane surface only from topo-
Digital Instruments NanoScope Scan size 500.0 nm Scan rate 0.2913 Hz Number of samples 256 Image Data Phase Data scale 10.00 ° Engage X Pos -19783.4 µm Engage Y Pos -42151.3 µm
view angle light angle
nm
400 300 200 100
x 100.000 nm/div z 10.000 °/div
0°
neuron_9_10_2004.021
Fig 10. Three-dimensional view of phase contrast imaging of the living cell membrane using TMAFM with scanning range of 500 nm. The image is obtained using a regular tip and then passing through the same bandpass filter as that used in Figure 9. However, there are no receptors can be identified in the image.
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Fig 11. A, AFM image of DNA ropes in its original shape. B, DNA ropes are cut by the AFM tip. The pushing force can be controlled to cut the DNA rope or only deform the DNA rope. The big scratches on the surface indicate strong pushing force applied, and small scratches imply a small pushing force. Arrows indicate the pushing directions.
phase angle is defined as the phase lag of the cantilever oscillation relative to the signal sent to the piezo driving the cantilever. Theoretic simulations and experiments of the cantilever dynamics in air have shown that phase contrast arises from differences in the energy dissipation between the tip and the sample. The phase shift is related analytically to the energy dissipated in the tip sample interaction, according to the following equation [26,27]: x A QED sinW ¼ þ x0 A0 pkAA0 where W is the phase angle, x/x0 is the working frequency/ resonance frequency, A/A 0 is the set-point amplitude/free amplitude, Q is the quality factor, E D is the energy dissipation, and k is the cantilever spring constant. The phase shift due to the tip-sample interaction, which involves energy dissipation, is the displacement of the noncontact solution to higher phase shifts and the intermittent contact solution to lower phase shift values. The more dissipative y C b A a
O
B
t x
Fig 12. Modeling deformation of DNA molecules and bundles under pushing by the AFM tip. Solid curve, DNA molecule (or DNA bundle) in its original status; dashed curve, new status of the DNA molecule. The DNA molecule (bundle) is pushed by the AFM tip from the start point O to the end point C. The DNA molecule (bundle) is only deformed within an affecting region from point A to B, which is bounded by a circle with radius of a; t is the thickness of the DNA molecule (bundle).
features will appear lighter in the noncontact regime, whereas they will appear darker in the intermittent contact regime [28]. When scanning the cell membrane surface using a tip functionalized with AT1 antibody, the tip-sample interaction force will increase when the tip closes to the AT1 receptor, resulting in a significant change of the phase shift. Because the topographic information is also convoluted to the phase contrast image but at a low frequency, a bandpass filter can be used to remove the low-frequency topographic information and high-frequency noise. After filtering the phase contrast image, only receptor images will be left on the surface. Figure 9 shows a phase contrast image of a neuron membrane obtained using the functionalized tip and then processed with a band-pass filter. The AT1 receptors are clearly indicated on the image. By using the optical scope equipped with the AFM system, the same cell can be scanned using a regular tip without any functionalization. By using the same band-pass filter to process the phase contrast image obtained with the regular tip, no receptor can be identified on the surface Figure 10. These experimental results show that single biomolecules like the receptors on the cell membrane surface can be recognized using the biologically functionalized tip. Manipulation of single DNA molecules using augmented reality system Because multishaped DNA molecules may have diverse properties, artificially modifying the molecules is necessary in the study of DNA properties. Kinks and deformation of DNA molecules can be created artificially using the AFMbased nanomanipulation system by controlling the pushing force between the tip and sample surface. The DNA molecules or DNA bundles can be either broken or deformed (Figure 11). A large pushing force in the normal direction usually breaks the DNA molecule, and a small pushing
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A
B
3.00 µm
0 Data type Z range
Height 50.00 nm
C
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itself, and f can be a fourth-order polynomial or a hyperbolic cosine function. In order to display the DNA deformation in real time, a local coordinate system is defined such that y-axis is along the pushing direction. The original shape of DNA inside the effect circle can be removed and then the new shape of DNA can be displayed with respect to the local coordinate system. By using the model developed in this section, manipulation of DNA molecules can be displayed in real time in the augmented reality environment. An example of DNA manipulation is shown in Figure 13, in which Figure 13, A, shows the DNA molecules in their original shapes, Figure 13, B, shows the manipulation of DNA molecules displayed in the augmented reality environment, and Figure 13, C, shows an AFM image after manipulation. It can be seen that several kinks have been created by slightly pushing the DNA molecules or bundles, and the kinks created in the augmented reality environment are relatively identical to the real results. Conclusions
0 Data type Z range
3.00 µm Height 50.00 nm
Fig 13. Pushing DNA on a polycarbonate surface (scanning range of 3 Am). A, Image of DNA before pushing. B, Real-time display on the augmented reality during pushing. C, A new scanning image after several pushing operations.
force may only deform the DNA molecule without breaking it. In Figure 11, B, the big scratches on the surface indicate a substantial pushing force applied on the AFM tip, and small scratches imply a small pushing force. The DNA bundle was broken when a big pushing force was applied but only deformed when a small pushing force was applied. To display in real time the deformation of DNA molecules or bundles in the augmented reality environment of the nanorobotic manipulation system, the deformation model of DNA molecules and bundles has to be identified. Although some forces dominant at the nanoscale level can be theoretically calculated, it is not feasible to compute them because some required model parameters are not available. Therefore, instead of using mathematic models for the force deformation of DNA molecules, an empirical method is adopted in this research. As shown in Figure 12, the DNA molecule (bundle) is pushed by the AFM tip from the start point O to the end point C. The DNA molecule (bundle) is only deformed within an affecting region from point A to B, which is bounded by a circle with radius of a. The radius is determined by the following empirical function, a = f(b,t), where b is the pushing distance before DNA broken, t is the thickness of DNA
The technique of using a functionalized tip to measure the interaction force between ligands and receptors by AFM has been discussed for more than a decade. Single-molecule recognition on well-prepared samples using a functionalized tip is also achieved in recent research. However, none of these techniques make possible the recognition of single molecules directly from a cell membrane surface. In this article, single AT1 receptor recognition directly from the cell membrane surface is achieved by use of an AFM tip biologically functionalized with the AT1 antibody. By passing the phase images into a bandpass filter, single receptor molecules are clearly identified in the processed image. Using this technique, further study of the trafficking behavior of the AT1 receptor is possible. In this article, an AFM-based nanorobotic system is also introduced, which can be operated both in the air and liquid conditions. Because the single receptor molecules can be identified in their physiologic condition, manipulation of single molecules on a living cell membrane surface also becomes possible. Our future research will show the efficiency of the proposed AFM-based nanorobotic system on manipulating single micromolecules such as receptor proteins in physiologic conditions. The controlled manipulation of selected molecules by AFM combined with high-resolution images will become a powerful approach to gaining information on the molecular interactions occurring within a biomolecular assembly or between biomolecular assemblies, thereby revealing the functionality of an individual biomolecule. We anticipate that the research that seeks to understand and exploit the interaction forces between nanoprobing mechanisms will provide a leap forward for biomedical research, whose progress is limited by the cumbersome and static multistep methods currently available.
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