basic research in situ sensing and manipulation of ... · basic research in situ sensing and...

Nanomedicine: Nanotechnology, B

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

aCollege of Engineering, Department of Electrical and Computer Engineering, Michigan State University, East Lansing, MI 48824bDepartment 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

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doi:10.1016/j.nano.20

T Corresponding

E-mail address: x

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

nt matter D 2005 Published by Elsevier Inc.

04.11.005

author.

[email protected] (N. Xi).

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 circum-

stances, 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

iology, and Medicine 1 (2005) 31–40

EnvironmentModel

AFM SystemUpdated AFM Image

InteractiveForce

Local Scan

Command

Generator

Haptic

Feedback

++Augmented Reality

Environment

Fig 1. AFM-based nanorobotic system, which includes an AFM system (nanorobot) and the augmented reality environment (visual haptic interface and

command generator).

G. Li et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 31–4032

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 well-

prepared 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 real-

time 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 manip-

ulation 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 subsys-

tems: 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 manipu-

lation. 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

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.

G. Li et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 1 (2005) 31–40 33

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 per-

formed 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 (APrM-

DEOS) (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 anti-

bodies. 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

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, Sigma-

Aldrich) by evaporating APTES and N,N-diisopropylethyl-

Fig 4. Low-magnification image of the living cells using the TMAFM with scanning range of 25 Am. Left, Height image. Right, Phase image.

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 SATP-

labeled 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,

Digital Instruments NanoScope

Engage X PosData scale

Engage Y Pos

Image DataNumber of samplesScan rateScan size 1.000 µm

0.2913 Hz256

Phase20.00 °-19783.4 µm-42151.3 µm

view anglelight angle

µm

0.8

0.6

0.4

0.20 °

x 0.200 µm/divz 20.000 °/div

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% heat-

inactivated horse serum (Hyclone, Logan, Utah). Cells were

then seeded in a 12-well culture plate with polyornithine-

coated glass coverslides inside. The cells were cultured in a

humid incubator at 378C with 5% carbon dioxide and 9% air.

Fig 6. The AFM image of living neuron cells with scanning rang

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 double-

helix 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,

of 90 Am. Left, Topographic image. Right, Phase image.

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

Fig 8. Final result of the cutting operation obtained from AFM image with

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 environ-

ments 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.

scanning range of 90 Am. Left, Height image. Right, Phase image.

Scan size 500.0 nm

Scan rate

Digital Instruments NanoScope

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


nm

400

300

200

100 x 100.000 nm/divz 20.000 °/div

neuron_9_10_2004.008_1

0 °

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

It is well known that recognizing specific proteins such as

receptors on the cell membrane surface only from topo-

200

100

neuron_9_10

Fig 10. Three-dimensional view of phase contrast imaging of the living cell memb

using a regular tip and then passing through the same bandpass filter as that used

graphic information is difficult. Fortunately, the new

technique, tapping-mode phase imaging, provides a promis-

ing solution. It can differentiate between areas with various

properties regardless of their topographic nature [26,27]. The

Digital Instruments NanoScopeScan size 500.0 nmScan rate 0.2913 HzNumber of samples 256Image Data PhaseData scale 10.00 °Engage X Pos -19783.4 µmEngage Y Pos -42151.3 µm


0 °

nm

400

300

x 100.000 nm/divz 10.000 °/div

_2004.021

rane using TMAFM with scanning range of 500 nm. The image is obtained

in Figure 9. However, there are no receptors can be identified in the image.

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]:

sinW ¼ xx0

A

A0

�þ QED

pkAA0

�

where W is the phase angle, x/x0 is the working frequency/

resonance frequency, A/A0 is the set-point amplitude/free

amplitude, Q is the quality factor, ED 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

t

a

b

y

x

B

O

C

A

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 informa-

tion 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 AFM-

based nanomanipulation system by controlling the pushing

force between the tip and sample surface. The DNA mo-

lecules 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

0 3.00 µm

0 3.00 µmData typeZ range

Height50.00 nm

Data typeZ range

Height50.00 nm

A B

C

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

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, manipu-

lation 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

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, manipu-

lation of single molecules on a living cell membrane

surface also becomes possible. Our future research will

show the efficiency of the proposed AFM-based nano-

robotic 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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