Scope of atomic force microscopy in the advancement of nanomedicinenopr.niscair.res.in/bitstream/123456789/10347/1/IJEB 48(10) 1020... · Indian Journal of Experimental Biology Vol

Indian Journal of Experimental Biology

Vol. 48, October 2010, pp. 1020-1036

Mini Review

Scope of atomic force microscopy in the advancement of nanomedicine

Srinivasan Ramachandran* & Ratnesh Lal*

Departments of Bioengineering, and Mechanical and Aerospace Engineering

University of California at San Diego, La Jolla, CA, 92093-0412 USA

One of the most exciting fields of current research is nanomedicine, but its definition and landscape remains elusive

due to its continuous expansion in all directions and thus constantly eroding its boundaries and defying definitions. This lack

of conceptual framework and confusing definitions was a hurdle for policy makers to enunciate credible goals and allocate

resources for the advancement of the field. In this mini review, we have provided a broad framework of nanomedicine

which defines its elusive landscape, and we hope this framework will accommodate its explosive growth in the future. Also,

we have highlighted the role and scope of atomic force microscopy techniques in the advancement of nanomedicine. For

improving health care of all that eventually would require successful intervention at fundamental biological processes, the

importance of understanding the structure-function relationship of biomolecules cannot be over emphasized. In this context,

AFM and its variants play a pivotal role in contributing towards the nanomedicine knowledge-base that is required for

fruitful developments in nano-diagnostics and nano-therapeutics.

Keywords: Atomic force microscopy (AFM), Nanoengineering, Nanomedicine, Nanoscience, Nanotechnology

The National Nanotechnology Initiative (NNI) has

defined nanotechnology as: “Nanotechnology is the

understanding and control of matter at dimensions

between approximately 1 and 100 nanometers

(nanoscale), where unique phenomena enable novel

applications. Encompassing nanoscale science,

engineering, and technology, nanotechnology involves

imaging, measuring, modeling, and manipulating matter

at this length scale”1. Unusual physical, chemical, and

biological properties can emerge in materials at the

nanoscale. These properties may differ in important

ways from the properties of bulk materials and single

atoms or molecules. However, the term ‘Nanomedicine’

has attracted and continues to attract several definitions

by various researchers/task forces2-6

at different points of

time. These confusing definitions and lack of conceptual

framework for Nanomedicine is even considered as a

stumbling block for the sustained growth and credibility

of this emerging field7-11

.

Definition of nanomedicine

From our point of view, we define Nanomedicine

as “branch of science that aims to understand and

intervene in at fundamental biological processes at

their native scale (nanoscale) using nanotechnology

towards delivering better health care for one and all”.

We believe this definition is broad-based that

encompasses utilization of nanotechnology (NNI’s

definition of nanotechnology) to: (a) understand

fundamental biologic processes in health to develop

better strategies to: prevent diseases, maintain and

promote positive health and (b) diagnose and restore

health/capacity of non-physiological conditions,

(through diagnostics, therapeutics and rehabilitation)

to provide better health care for an individual as well

as to the community as a whole. This framework

could accommodate natural expansion of this exciting

field in the future. A pictorial representation of the

nanomedicine system, its components and their

relationship is presented in Fig. 1.

Understanding the size dependent properties of

matter at nanoscale in physics, chemistry, biology and

other cross-disciplines (nanoscience) would form the

primary source of knowledge for the development of

nanoengineering and technology. In figure 1, this

layer is depicted at the bottom of the pyramid. The

inputs from the nanoscientists would enable researchers

to engineer man made materials/tools/devices/

techniques/methods at nanoscale or macroscopic

systems to study nanoscale events for systematic

interrogation of natural processes in the body. In this

process, nanoengineers and technologists would learn

from biological systems how it synthesizes/

assembles/renews nanostructures and that would

_____________

*Co-correspondent authors

Telephone: 858-822-1322

Fax: 858-822-3976

E-mail: [email protected]; [email protected]

RAMACHANDRAN & LAL: ATOMIC FORCE MICROSCOPY & NANOMEDICINE

1021

immensely help to develop newer nanotechnologies.

Similarly, advancements in nanoengineering and

technology would provide appropriate newer tools to

nanoscientists for further understand the basis of size

dependent properties of matter. This forms the

intermediate layer of nanomedicine pyramid and the

information flows bidirectional. Ultimately, these

tools would be applied in medicine (top layer) to

understand normal and abnormal processes happening

in the body at their native scale. The continuous

exchange of information between the bottom two

layers would constantly evolve newer and better tools

to study nanomedicine (Fig. 1). This would help to

achieve the goals of nanomedicine both at an

individual level (personalized medicine) and at global

level for the whole community.

Why nanomedicine? Limitations of the current

medical system The current system of medical practice is mostly

based on ensemble observations from macroscopic

phenomena obtained from in vitro cell models, animal

models, inanimate microscopic studies, population

studies (epidemiological), simulations etc. These data

are far from adequate and doesn’t reflect the actual

process at molecular resolution, which translates into

poor/misunderstanding of health and disease.

Understanding the molecular pathogenesis of a

disease is the sine qua non for developing successful

diagnostics and therapeutics. For example, interaction

of drug molecules with human body at molecular

level is essential to minimize/prevent drug induced

side effects, drug interactions and toxicity. Therefore

the need to develop nanotechnologies to study and

affect the biological processes at molecular resolution

is critical to advance nanomedicine.

Current landscape of nanomedicine The continued big bang like expansion of

nanoscience and technology research in every field

in the last decade is constantly pushing the frontiers

of nanomedicine thus making it to difficult to define

the landscape of nanomedicine that is insensitive to

time component. Several attempts were made in the

past to classify nanotechnologies employed in

nanomedicine3,12-14

. Here we outline the scope of

nanomedicine that is insensitive to time (Fig. 2).

Broadly, they could be classified based on their

complexity as: (1) tools/devices (2) techniques/

methods. Each of their application to nanomedicine

could be further divided into areas of contribution to

(a) knowledgebase of nanomedicine—towards

comprehensive understanding of molecular

medicine, (b) therapeutics, (c) diagnostics and

(d) prevention of disease/promotion of

health/restoring the capacity (rehabilitation)/policy

making etc., wherever applicable. Listing all the

probable candidates under each section is beyond the

scope of this article but few key tools and techniques

were listed under them.

Basic tools (devices): A tool is defined as “a device

or a piece of equipment that typically provides a

mechanical advantage in accomplishing a task or

enables the accomplishment of a task not otherwise

possible”15,16

. On the other hand, a technique is

defined as “the systematic procedure by which a

complex or scientific task is accomplished17

”, also “a

practical method, skill, or art applied to a particular

task”18

. In simpler terms, a tool/device is a means of

execution of a task and a technique/method is a

systematic way of solving a complex problem often

with tools. Using this framework, one could classify

nanotechnologies into tools/devices and techniques/

methods. In more specific terms, any tool that helps to

observe, measure, manipulate, modify and affect

structure at nanoscale (imaging tools) including but

not limited to AFM and its variants, EM and its

variants, Single molecule fluorescence tools, optical/

magnetic tweezers, nanomanipulators; nanoscale

materials which includes both incidental and

engineered materials such as nanoparticles,

nanofibers, nanoshells, nanofilms, biosensors, etc.,

belong to the class of tools/devices.

Techniques/methods: These are more complex in

their structural architecture than tools/devices and

they are utilized to achieve solution(s) to a

problem through a systematic approach. This includes

Fig. 1—Components of nanomedicine and their inter-relationship

towards better and positive health care for all

INDIAN J EXP BOIL, OCTOBER 2010

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wide array of techniques to develop toolkits, cross

technology platforms, large scale manufacturing

methods, computer programs etc., which helps to

further study, measure, manipulate, modify and affect

biological processes at nanoscale. For example,

(i) application of a tool (fluorescently labeled

nanoparticles) to understand specific molecular

interactions; (ii) combining several layers of

tools/devices to develop a system for a specific task

like multiplexing sensors, actuators and controllers

integrated on fluidic devices for automated high

throughput screening and diagnostic applications;

(iii) industrial manufacturing techniques for

nanostructured materials and devices;

(iv) high throughput array techniques; (v)

bioinformatics; (vi) systems biology; (vii) molecular

dynamic simulations, etc.

Application of these tools and techniques to

nanomedicine could be broadly classified into

(a) expanding molecular medicine (nanomedicine)

knowledge-base, (b) prevention and rehabilitation,

(c) therapeutics, and (d) diagnostics

(a) Molecular medicine (nanomedicine)

knowledge-baseAs mentioned before,

understanding health and disease at molecular

resolution (nanoscale) is vital for developing any

successful diagnostics or therapeutic measures against

any illness. This knowledge would help the health

care providers, policy makers and alike to devise

strategic plans to achieve health for all both at

personalized level and at global level.

(b) Disease prevention/health promotion/restoring

capacity (rehabilitation)The most cost effective

way to achieve health for all is to prevent diseases and

promote health in healthy individuals through health

education. Information from the knowledgebase

would help to achieve this. Similarly, restoring the

capacity (rehabilitation) of individuals for whom

curative treatment has little or limited role is

important for improving their quality of life. This

could be achieved through utilization of nano devices

and nanostructured materials in tissue/organ

assistance and replacement; artificial tissues/

organs/implants to restore capacity due to disease or

injury.

(c) NanotherapeuticsApplication of the above

tools and techniques that affects at molecular level to

promote health and prevent/cure/ameliorate disease is

called nanotherapeutics. Examples: nano-

pharmaceuticals, smart drug delivery systems,

nanostructured materials, nanodevices in medicine

and surgery, etc.

(d) NanodiagnosticsApplication of tools and

techniques to assess and monitor clinical parameters

Fig. 2—Classification of nanotechnologies in medicine. To emphasize the ever growing field and often overlapping relationship among

its components, they are represented in radial venn diagram


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at molecular level that helps in diagnosis of health and

disease is called nanodiagnostics. A few noteworthy

examples includes: nanosensors, nanoradio-

pharmaceuticals, nanoarray, nanochip, nanofluidics,

nanopore, nanoparticle, nanovesicle based techniques

for monitoring DNA/RNA/protein/biomolecules/

chemicals etc. However, given the significance of

understanding the structure-function relationship of

biomolecules which is critical for the contribution to

the nanomedicine knowledgebase upon which further

developments in nano-diagnostics and nano-

therapeutics ultimately depends, this article aims to

review the imaging tools and techniques, AFM in

particular that reveal structure-function of biological

structures at nanoscale in their native (near native)

physiological environments.

Atomic force microscopy in nanomedicine

Atomic force microscopy (AFM) is a type of

scanning probe microscopy (SPM) technique

developed by G Binnig, CF Quate and C Gerber in

198619

. It is one of the most important tools in

nanoscience and technology to visualize, manipulate

and modify single biomolecules at their near native

environment. Considerable advances in biomedical

research were gained through structural biology

studies using electron microscopy, X-ray diffraction,

nuclear magnetic resonance etc. However, their main

limitations being extensive sample preparations,

crystallization problems, size limitations and most

importantly their non-physiological imaging

environments doesn’t provide any functional

information of the samples analyzed. On the other

hand, AFM doesn’t require such elaborate sample

preparations; it allows examining the samples at their

physiological conditions thus permit functional and

dynamic studies under noninvasive conditions. Most

importantly, it allows easy integration of other

complementary modalities like fluorescence microscopy

(including widefield, confocal, TIRF, FRET, etc.),

electrophysiology20

, optical tweezers, microfluidics, etc.,

thus providing a powerful platform to obtain structure-

function data at single molecular level.

In its simplest form, AFM has a cantilever with a

sharp probe at its tip to scan the sample surface. When

the tip is drawn close to a surface, depending upon the

surface characteristics, the tip experiences a variety of

force interactions that deflects the cantilever which is

detected by a laser spot (most commonly) on a

quadrant photodiode. A feedback control maintains

the tip-sample separation via cantilever deflection to

maintain the constant force experienced by the tip. In

certain configurations, the sample is mounted on a

X,Y,Z piezoelectric tube that can move the sample in

z axis to maintain the same force experienced by the

tip and move the sample in X,Y to enable raster scan

across the sample. By doing so, it captures the

topography of the sample down to molecular/atomic

resolutions (0.1 nm in Z and 1 nm in X,Y) (Fig. 3)21

.

There are two main imaging modes of AFM:

(1) contact mode, in which an electronic feedback

circuit maintains a constant deflection, ensuring a

constant force of interaction between tip and sample.

The amount of ‘z’ variation needed to maintain the

constant interaction force is plotted versus the x and y

coordinates, producing a topographic image and

(2) tapping mode, in which the amplitude of vibration

of an oscillating cantilever is maintained constant

during scanning. In the tapping mode, the phase lag

between the driving circuit and the actual tip vibration

is also measured. The deflection of the cantilever in

the contact mode and the damping of vibration

amplitude in tapping mode are caused by a sum of

attractive and repulsive forces.

The dominant repulsive force sensed by the AFM

cantilever results from the overlapping of electron

orbitals between the atoms of the tip and of the

sample. The dominant attractive force is Van der

Waals interaction, which is primarily due to non

localized dipole-dipole interactions. Another strong

attractive force component that exists while imaging

in air is the meniscus-surface force due to adsorbed

water layers. In fluids, consideration should be given

Fig. 3—Schematic of the operating principle of AFM (modified

from Wikipedia21)


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to electrostatic interactions between charges and the

sample and tip, and structural forces such as hydration

force, solvation forces, and adhesion forces, see22,23

.

In the tapping mode, conductive/magnetically coated

cantilevers can sense electrostatic and magnetic

forces, and image for instance magnetic domains,

surface charge distributions, local surface capacitance

and local conductance. These forces can be used to

generate images that provide valuable information on

the differences in local surface chemistry, like

separate lipid and protein clusters in a membrane, and

as such can be used as effective sensors of energy and

the functional states of a specimen.

There are two major types of AFM’s for biological

applications: (a) multimode AFM, which offers

multiple SPM modes, including AFM,

electrochemical AFM (ECAFM), scanning tunneling

microscopy (STM), electrochemical STM (ECSTM)

and tapping mode. If offers the maximal resolution in

atomic scales, and the other type is (b) bio-scope

AFM which integrates the best of optical microscopy

and AFM to help life scientists explore new frontiers.

The ability of the AFM to create 3D surface

topography with resolution down to 0.1-0.2 nm has

made it an essential tool for imaging surfaces in

applications ranging from material science to cell

biology. As the original AFM designed was for

material science applications24

it couldn’t be used for

imaging biological specimens in their native

environment (in buffer solutions). A major

breakthrough in biological applications of AFM

occurred with the invention of liquid cell by Paul

Hansma’s lab at UC Santa Barbara in 198925,26

, and

the next breakthrough came with tapping mode in

liquid by Putman et al27

. Since then AFM has been

used in variety of biological applications ranging from

high resolution imaging of membrane protein

crystals28-30

, imaging molecular complexes like

nucleosomes31

, imaging and mapping molecular

interaction sites example fibronectin and heparin32

,

visualizing the action of enzymes33

, real time imaging

of single molecule degradation e.g. collagen

proteolysis34,35

, mapping of antigen-antibody

interactions36

, soft tissue/cell imaging and their

mechanical properties37

, imaging ion channels38

,

manipulating single molecules39

, etc.

It is difficult to classify the applications of AFM in

nanomedicine in an orderly fashion due to their

exhaustive list of applications and overlapping nature

into several areas. But it could be broadly classified

into (a) tools and techniques based on AFM system,

and (b) cantilever based applications. As a tool, AFM

was widely used to image and measure sample

parameters like size, shape, distribution etc.; as a

technique it was used as a tool to develop

methods/applications to address complex issues. For

example, to study interactions between molecules

(antigen-antibody, receptor-ligand, protein-protein,

protein-DNA etc.) and measure their interaction forces,

study protein folding, study structure and function of

ion channels, membrane proteins, analyze mechanical

properties of cells and its organelles, etc., AFM

cantilevers are exploited as quantitative nanosensor for

several applications due to their extreme sensitivity and

functionalization opportunities, that makes it a versatile

tool for diverse applications including viscosity sensor,

detection of complementary structures, etc.

For the sake of simplicity we have grouped the

AFM applications into the following:

Imaging applications

Single biomoleculesThere are several reviews that

cover single biomolecular imaging with AFM40-44

. It is

the first time that biologists were able to visualize

single biomolecules in their native environment.

Following are some of the important milestones in this

area. Lindsay et al.45

achieved the first AFM image of

DNA in water, Vesenka et al.46

improved the resolution

of DNA by imaging them on mica, further

improvement came by changing the imaging liquid47

measurements of nucleosome48

and imaging of DNA-

protein complexes by Bustamante et al.49

and

Lyubchenko et al50

.

Membrane proteinBacteriorhodopsin was the first

membrane protein imaged with AFM51,52

, the images

clearly showed periodic structures (6.2 nm) between the

units. Lal and Hoh studied the gap junctions proteins and

applied imaging force to dissect one half of the plasma

membrane to reveal the extracellular face of the hemi-

gapjunctions for the first time53,54

. This study

exemplifies that AFM could be used to manipulate

biological specimens at the nanoscale. Lal et al. obtained

3D crystal structure of porin channels30,55

which revealed

the subunit organization and channel pore which

correlated with other ultrastructural studies.

Cell membraneDefining cell membrane

structures and their changes could be used as marker

for diseased versus normal. The identification

membrane-associated targets, such as ion channels

and receptors, is complex and mostly indirect since

these channels, receptors, and other nanoscale


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structures are smaller than the current resolution of light

microscopic imaging. In one of the earliest examples of

AFM mapping of cell membrane receptors, the AFM

was used to identify the individual nicotinic

acetylcholine receptor (nAchR) that was expressed in

Xenopus oocytes56,57

. Using ion conductance

microscopy, Hansma et al.58

, Proksch et al.59

, and

intermolecular force mapping, AFM can provide

density, distribution, clustering, and functional viability

of most of the cell membrane macromolecules.

Force spectroscopyIntermolecular interactions

and defining effectors: AFM has developed into a

valuable tool for measuring molecular interactions.

The possibility of linking molecules to the probing tip

provides a range of options to probe molecular

interactions between the probe and the sample surface

to study the interaction and quantify the force of

interaction between them60,36,61-63,39

. Using such

functionalized cantilevers, adhesion forces have been

measured and mapped between receptors and ligands

on the surface of living cells (Fig. 4).

The regional distribution as well as ligand or

antibody induced clustering of VEGF receptors have

been reported (Fig. 5) by Almqvist et al65

. Using

combined AFM with fluorescence microscopy, Quist

et al.66

, have examined cell volume regulation in

response to external perturbations. The added benefit

of force mapping is the simultaneous mapping of the

stiffness of the cell membrane (Fig. 6), thus AFM

cantilevers can be used as “stiffness sensors.” There

are active efforts now to use “stiffness sensors” to

distinguish cancerous versus normal cells by Cross

et al67

.

Intermolecular interaction force can also be used to

sense normal versus abnormally folded proteins that

underlie many diseases. As an example, Liu et al.39

,

used an AFM tip conjugated with antibodies

specific to different regions of a hemichannel

peptide, connexin 43 (Cx43), to map the specific

Cx43 epitopes that open and close the hemichannel

in response to changing calcium concentration

(Fig. 7).

Fig. 4—Left panels: Adhesion forces between VEGF receptor and antibody (A) with distribution of unbinding forces (B) blocking

peptide prevents the interaction (C) and (D) show tapping mode image of VEGF receptors on mica. (E) and (F) show adhesion forces

between the VEGF receptor on endothelial cells and antibody on the tip without (E) and with (F) the presence of a blocking peptide.

Right panels: AFM images of endothelial cells before (A) and after (B) addition of VEGF, showing cytoskeletal reorganization.

Fluorescence images showing the presence of Flk-1 receptors (C, D) and control (E) with nonspecific antibody [reproduced from Jai

Raman et al.64 and Almqvist et al.65]


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The force required to unwind the peptide

(for channel opening) was correlated with the

mobility of specific portions of Cx43. The precise

estimate of the energy to unfold and stretch (contract)

peptides using the AFM shows the promise of using

AFM for more accurate indication of the therapeutic

efficacy of drugs and pharmacological agents with

single molecule resolution.

Ramachandran et al.68

used AFM to characterize

cisplatin loaded nanoliposomes for their size, volume,

elasticity measurements. They showed cisplatin

encapsulated nanoliposomes had higher stiffness and

stability compared to their controls. They combined

AFM with fluorescence microscopy to show that

cisplatin laded nanoliposomes were efficiently taken

up by the cells resulting in their death. This work

showcased that AFM could be used for evaluating the

pharmaceutical preparations as well.

Imaging ion channels on reconstituted lipid bilayers

Membrane proteins that span the plasma membrane

several times are the most difficult ones to isolate and

study using conventional high resolution techniques

like NMR, EM etc. But some of them are amenable to

isolation but difficult to crystallize them69

. Under

these circumstances, AFM will be an important tool to

study them under physiological conditions70

. One

such example is application of AFM to reveal the

structure of connexin43. Thimm et al.23

showed the

subunit architecture and open-closed conformation of

Fig. 5—Top histograms show distribution of adhesion forces between the anti-VEGFR antibody-conjugated AFM tip and VEGFR in the

cell plasma membrane. Middle and bottom panels show simultaneous acquisition of topography (middle panel) and elasticity images

(bottom panel) before, and 10 and 45 minutes after adding VEGF antibody. Clustering of receptors can be observed both in topography,

and elasticity maps (spots labeled 1-4). Reproduced from Jai Raman et al.64 ; for details see Almqvist et al.65]


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Fig. 6—Effect of extracellular calcium removal on cell elasticity; and effect of cytochalasin D on cell volume and structure. [Reproduced

from Quist et al.66]

Fig. 7—AFM antibody conjugated tip pulling experiment: Sensing intermolecular interaction for mapping conformational epitopes. Left panel:

Model of a gap junction plaque with opposing cell membranes with hexagonally packed hemichannels (left). Schematic of force spectroscopy

measurement showing binding of antibody connected to the AFM tip with flexible PEG spacers to Cx43 hemichannels reconstituted in the lipid

bilayer (middle) and the schematic of Cx43 membrane topology with two extracellular loops, one cytoplasmic loop, and the cytoplasmic

carboxyl-terminal domain and locations of antigenic binding sites for anti-CT252–270 and anti-CT360–382 (right) are shown. Right panel:

Probability histograms of the rupture forces of the measured anti-CT252–270-Cx43 (A), anti-CT360–382-Cx43 (B), and GAP26-Cx43

interactions (C). D: a representative force-extension curve showing specific avidin-biotin interaction in the PEG spacer extension test system. E:

the average extension of PEG spacer stretching (~28 nm). F: histograms of measured tether extensions in anti-CT252–270-Cx43, anti-CT360–

382-Cx43, and GAP26-Cx43, respectively. [Reproduced from Jai Raman et al.64; for details see Liu et al.39]


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connexin43 to extracellular calcium variation (Fig. 8).

This study could be extended to any other proteins in

principle. Similarly, isolated peptides/proteins/ion-

channels/receptors/etc., could be reconstituted in lipid

bilayers and imaged with AFM. Lal’s group has

imaged several amyloid peptides Aβ1-40/42, A-Dan,

A-Bri, Amylin, α-synuclein, SAA (Fig. 9), K3 and

non-amyloidogenic peptides on lipid bilayers to show

that they form ion-channel like structures38,71-75

.

Cell mechanics The proper functioning of cells depends on

controlled biochemical processes as well as regulated

cytoskeletal structural reorganization. Reorganization

of the cytoskeleton and changes in its mechanical

properties play key roles in cell growth, migration,

and development76

. The local mechanical properties

of a cell are closely associated with biochemical

gradients across the membrane, but most techniques

that have been used to study single cells average over

the entire cell77

. AFM gives the opportunity to

measure the mechanical properties of cells with high

spatial resolution. For instance, rat atrial myocytes

were imaged78,79

clearly showing the cytoskeletal

network beneath the cell membrane and myofibrillar

structure). Using a constant cantilever deflection

maintained by feedback, contractile activity and the

change in contractile activity using perturbations in

the buffer environment was examined. This

demonstrates the possibility to quantify the coupling

between subcellular substrates to cellular functions

such as contraction, migration, growth, and

differentiation.

Lal and his colleagues80,81

examined the endothelial

barrier permeability using AFM force measurements

on human pulmonary microvascular endothelial cells.

Also, force spectroscopy measurements were applied

to study the structural and mechanical properties of

bacterial biofilms82

. Such monitoring of cell

mechanics can be used effectively to diagnose

pathologies associated with abnormal cell mechanics

as well as to monitor the efficacy of therapeutics that

are expected to correct abnormal cell mechanics.

Tissue nanostructure and property

Biological fibers have nanomechanical properties

that depend on their morphology as well as the

chemical heterogeneity of their constituent subunits.

A detailed understanding of tissue elasticity can be

used for efficient and early diagnosis and for

monitoring the progression of diseases and their

treatments, especially diseases of bones, calcified

tissues and carcinoma. Indeed, there are some new

diagnostic tools being tested to diagnose bone

diseases83,84

, that resulted from our understanding of

the tissue mechanics at the molecular level85

. The

Fig. 8— A–G: 3D height images of individual connexons imaged in buffer with varying concentrations of calcium. Reproduced from

Thimm et al.23


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correlation between the mechanical properties and the

heterogeneous subunits on a nano scale has been

limited. Using AFM, such correlation studies are

made possible by Parbhu et al.86

An example of such

studies is summarized in Fig. 10. AFM force mapping

was used to study the mechanical properties of the

different constituents of wool fibers. It was shown

that the exo-cuticle part has the highest elastic

modulus while the endo-cuticle and cortical regions of

the fiber have significantly lower modulus. The

indentations made by a diamond tipped cantilever

look indeed distinctly different for the different

regions (Fig.10, right panel). Furthermore, the AFM

study could give conclusive evidence of the role of

disulfide bonds in the fiber stiffness. Reduction of

such bonds, abundant in the exo-cuticle, using DTT,

resulted in a reduction of modulus.

Using the unique feature of defined

nanoindentation, one can create specific patterned

structures, like nanocavities, nanopores, nanowells

and nanochannels and use them for array sensors of

pathogens and toxicants.

Multimodal imaging platform

AFM and TIRF microscopy—The architecture of

Bioscope AFM allows one to integrate it with optical

microscopy, electrophysiology, electro-optical

tweezers etc to obtain complementary imaging

information. One such example is Ramachandran

et al.87

who integrated the bioscope AFM with a novel

LED based TIRF microscopy system to study

molecular permeability through single reconstituted

hemichannel suspended over a nanopore.

AFM and ion conductance/permeability assay

tools—Combining an AFM with other tools is

important for obtaining simultaneous information

about sample functional properties and activity. In its

earliest form of a combined AFM and an ionic

conductance measurement system, a nanometer inner

diameter glass electrode served two purposes; it acted

like an AFM cantilevered tip and also an electrode for

recording ionic conductance58

. In a later version,

using appropriate voltage drop across a bilayer/cell

membrane, conductance through pores in a synthetic

nuclear filter was measured59

.

As an extension of the combined AFM and ion

conductance measuring system, AFM has recently

been combined with advanced nanochip supported

double chamber permeability and a transport assay

system (Fig. 11). This combined system allows study

of the activity of ion channels and pores and that will

be useful for high throughput sensing of pathogens

and toxic signals that modify channel activity and in

turn can also be used to design antidotes (potential

Fig. 9—Various amyloid beta peptides form ion channel like structures when reconstituted and imaged with high resolution AFM

(adapted from Quist et al.38)


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therapeutics). Figure 11 shows an example of one

such design in which ionic conductance through

gramicidin channels is reconstituted in a lipid bilayer

supported over a silicon chip with nanopores ~ 70 nm

in diameter. Gramicidin is a gram negative bacteria

that induces ion channel-like activity when in contact

with the cell membrane, and is a good test structure

for defining membrane permeability and transport.

Significantly, this study also shows that the AFM

imaging force is soft enough to image delicate and

fragile biological membranes that could be used for

screening membrane modifiers (e.g., pathogens and

toxicants).

Ionescu et al.88

were able to implement a new

conducting AFM tip that can be used for direct study

of the conformational changes in ion channels as

would occur in response to pathogens/toxicants. It can

also be used for characterizing many advanced

materials with wide biomedical applications. In one

such study, they studied the direct structure-function

relation in conjugated polymer blends. Polymer

blends have wide biological applications, including

microactuators89

, chemical sensors90

, and light

emitting diodes91

. Conjugated optically and

electrically sensitive polymer blends are being tested

as sensors for pathological biological markers.

AFM cantilever based applications Emerging technologies are generating a myriad of

array sensors for high throughput screening of

samples (genome, transcriptome, proteome,

metabolome etc.) that rely on specific interactions

using antibodies, peptides, and fluorescent labels.

For identifying biomarkers of diseases (e.g.

channelopathies), assaying the efficacy of drugs and

screening drugs, two powerful avenues involve patch

Fig. 10—Left panels: schematic diagram of wool fiber (A). TEM image (B, higher magnification in (D)) and AFM image (C, higher

magnification in (E)) of same wool fiber region. AFM and TEM images correlate well with respect to identified subcellular structures.

Center panels: force curves on Cortex (top), Exo-cuticle (middle) and embedding resin (bottom). Solid line is collected on hard glass

surface. Larger shaded area indicates more elastic surface. Right panels: Top right image shows corresponding AFM image. Bottom right

image shows surface after indentation with diamond tip to create specific patterns on wool fiber. Reproduced from Jai Raman et al.64; for

details see Parbhu et al86.


1031

clamp(s) on chips and nano-micro-fluidics92-94

.

Significantly, AFM can be combined with both

techniques easily and thus provide a very powerful

platform for fast, reliable and highly sensitive high

throughput technique.

Nano-micro fluidic viscosity, velocity, and

molecular affinity sensor Parallel read out techniques based on AFM

cantilever arrays95,96

enable to develop a very high

sensitive, high throughput detection platform. When

combined with nano-microfluidic chambers, these

array nanosensors will become high throughput

screening in small volumes. They can probe for

instance multiple live cells for their elastic properties

or the presence of receptors in the cell membrane.

Similarly, coating a parallel array of cantilever with a

different material or reagent on each lever results in a

‘chemical nose’ that can sense a variety of chemicals

or toxins in very small volumes97

.

Fig. 11—AFM integrated with a nanochip-separated double chamber electrical recording system. A: AFM image of arrays of nanopores.

The pores are produced in a silicon nitride chip using electron beam lithography and range in size from 50 (top rows) to 100 nm (rows on

the right). B: After deposition of lipid bilayers, AFM imaging in PBS shows a bilayer covering the pores. The bilayer is not fully

contiguous and has several holes (red arrows); the cross section at a hole in the bilayer (inset) indicates the thickness of a lipid bilayer

~5.3 nm. Blue arrows indicate 500 nm wide Corner Alignment Marks, red arrows indicate defects in the bilayer. C: Schematic of the

liquid cell AFM setup for imaging the silicon nitride chip and bilayer. AFM images (panel D) and conductance maps (panel E) over a

pore in a micro-fabricated Silicon chip when the lipid bilayer is formed. F: When gramicidin is added to the bilayer, the overall current

across the pores increases and conductance increases from picoSiemens to nanoSiemens suggesting the formation of hundreds of

gramicidin ion channels. Reproduced from Jai Raman et al.64; for details, see Quist et al92.


1032

Microscale fluid velocity, viscosity, and shear

stress play significant roles in tissue sustenance and

several pathologies (e.g., atherosclerosis,

thrombosis). Also, local fluid mechanics would

affect interaction of any therapeutics with their

targets and would even be indicative of local

pathologies, such as reduced flow rate around an

occluded vein or artery. Yet we know very little

about them. Liquid viscosity is hard to measure

with high precision and in small volumes.

Traditionally, ultrasonic devices are used to

measure viscosity98

. They operate at MHz

frequency at which the viscosity of non-Newtonian

fluids can be different at low-frequencies which is

of greater clinical interest. Flexural-mode

resonance devices, such as microfabricated

cantilevers, may be more reliable since they allow

for measurement at lower frequencies. Using an

optical detection approach typical in standard AFM

equipment, viscous drag has been measured using a

piezoelectric actuator to vibrate an AFM silicon

cantilever99

. Other ways of using AFM to measure

liquid viscosity include measuring the torsion in an

AFM cantilever while scanning a whisker tip inside

the liquid100

.

Figure 12 shows schematics of cantilevered

microfluidic sensing tools in which a piezoelectric

cantilever senses viscosity and velocity of fluids with

different viscosity and ionic composition and can be

easily adapted for screening biomarkers in blood and

body fluids. When combined with local fluorescence

sensors (eg, SPR, NSOM, photo-sensitive

Fig. 12—Nanosensors for biomarkers and diagnostics. Microfluidic viscosity, velocity, molecular affinity sensors are shown. A:

Schematics of cantilevered microfluidic sensor. B: Setup of the stainless steel needle (top) and silicon channels (bottom). C: FIB-milled

cantilevers serve as flow and viscosity sensors for biological fluids. D: Comparison of voltage readout at different flow speeds..

Reproduced from Jai Raman et al.64; for details see Quist et al100.


1033

nanoparticles), electrical properties (e.g., piezoelectric

circuitry) and mechanical properties (e.g., cantilever

deflection), an array of cantilevers with specific

complements can provide a powerful and highly

sensitive tool for high throughput screening of

pathologies and diagnostics from a very small

(nano-micro litter) amounts of biofluids (Fig. 13).

Conclusions This mini review has provided an operational

definition and framework for nanomedicine, its goals

and scope. In particular, it emphasized the role of

AFM in the advancement of nanomedicine owing to

its unique advantages to see beyond resolution limits

of biological samples in their native environment. Its

versatility and flexibility to integrate with other

complementary techniques offers unique

opportunities to develop a powerful platform of

technologies to advance nanomedicine in its every

sphere (Fig. 2).

Acknowledgement This work was supported by the research grant

((7R01DA025296-03) from National Institute on

Drug Abuse (NIDA).

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32 Lin H, Lal R & Clegg DO, Imaging and mapping heparin-

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33 Radmacher M, Fritz M, Hansma HG & Hansma PK, Direct

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36 Hinterdorfer P, Baumgartner W, Gruber HJ, Schilcher K &

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1035

58 Hansma PK, Drake B, Marti O, Gould SAC & Prater CB,

The scanning ion-conductance microscope, Science, 243

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59 Proksch R, Lal R, Hansma PK, Morse D & Stucky G,

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60 Baumgartner W, Hinterdorfer P, Ness W, Raab A,

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the leukocyte function-associated antigen-1/intercellular

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64 Jai Raman NJ, Sungho Jin, & Lal Ratnesh, Nanoimaging

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Yun, (Artech House, Boston ), 2002, 2009.

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R, Elasticity and adhesion force mapping reveals real-time

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gap-junctional hemichannels: Extracellular calcium-

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67 Cross SE, Kreth J, Zhu L, Sullivan R, Shi WY, Qi FX &

Gimzewski JK, Nanomechanical properties of glucans and

associated cell-surface adhesion of Streptococcus mutans

probed by atomic force microscopy under in situ conditions,

Microbiology-Sgm, 153 (2007) 3124.

68 Ramachandran S, Quist AP, Kumar S & Lal R, Cisplatin

nanoliposomes for cancer therapy: AFM and fluorescence

Imaging of cisplatin encapsulation, stability, cellular uptake,

and toxicity, Langmuir, 22 (2006) 8156.

69 Kuhlbrandt W, 2-Dimensional crystallization of membrane-

proteins, Quarterly Rev Biophys, 25 (1992) 1.

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open state, Nature, 373(1995) 37.

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structure and relevance to amyloid channel paradigm,

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72 Lal R, Quist AP, Duodevski I & Lin H, Atomic force

microscopy of amyloid proteins: Examining real-time 3D

conformations and role in protein conformational diseases, J

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73 Rhee SK, Quist AP & Lal R, Imaging A beta P1-42-channel

structure and activity using atomic force &

immunofluorescence microscopy and Ca-45(2+) uptake

assay, Biophysical J,74 (1998) A315.

74 Rhee SK, Quist AP & Lal R, Amyloid beta protein-(1-42)

forms calcium-permeable, Zn2+-sensitive channel, J Biol

Chem, 273 (1998) 13379.

75 Mustata M, Capone R, Jang H, Arce FT, Ramachandran S,

Lal R & Nussinov R, K3 Fragment of amyloidogenic beta(2)-

microglobulin forms ion channels: Implication for dialysis

related amyloidosis, J Am Chem Soc, 131(2009) 14938.

76 Lal R, Drake B, Blumberg D, Saner DR, Hansma PK &

Feinstein SC, Imaging real-time neurite outgrowth and

cytoskeletal reorganization with an atomic-force

microscope, Am J Physiol-Cell Physiol 38(1995) C275.

77 Brady AJ, Mechanical-properties of isolated cardiac

myocytes, Physiological Rev, 71 (1991) 413.

78 Shroff SG, Saner DR & Lal R, Atomic-force microscopy of

atrial cells - local viscoelastic mechanical-properties and

imaging of cytoskeleton, Biophysical J, 66 (1994) A278.

79 Shroff SG, Saner DR & Lal R, Dynamic micromechanical

properties of cultured rat atrial myocytes measured by

atomic-force microscopy, Am J Physiol-Cell Physiol, 38

(1995) C286.

80 Arce FT, Whitlock JL, Birukova AA, Birukov KG, Arnsdorf

MF, Lal R, Garcia JGN & Dudek SM, Regulation of the

micromechanical properties of pulmonary endothelium by

S1P and thrombin: Role of cortactin, Biophysical J, 95

(2008) 886.

81 Birukova AA, Arce FT, Moldobaeva N, Dudek SM, Garcia

JGN, Lal R & Birukov KG, Endothelial permeability is

controlled by spatially defined cytoskeletal mechanics:

Atomic force microscopy force mapping of pulmonary

endothelial monolayer, Nanomed-Nanotech Biol Med, 5

(2009) 30.

82 Arce FT, Carlson R, Monds J, Veeh R, Hu FZ, Stewart PS,

Lal R, Ehrlich GD & Avci R, Nanoscale structural and

mechanical properties of nontypeable haemophilus

influenzae biofilms, J Bacteriol, 191 (2009) 2512.

83 Fantner GE, Birkedal H, Kindt JH, Hassenkam T, Weaver

JC, Cutroni JA, Bosma BL, Bawazer L, Finch MM, Cidade

GAG, Morse DE, Stucky GD & Hansma PK, Influence of

the degradation of the organic matrix on the microscopic

fracture behavior of trabecular bone, Bone, 35 (2004) 1013.

84 Fantner GE, Hassenkam T, Kindt JH, Weaver JC, Birkedal

H, Pechenik L, Cutroni JA, Cidade GAG, Stucky GD,

Morse DE & Hansma PK, Sacrificial bonds and hidden

length dissipate energy as mineralized fibrils separate during

bone fracture, Nature Materials, 4 (2005) 12.

85 Thompson JB, Kindt JH, Drake B, Hansma HG, Morse DE

& Hansma PK, Bone indentation recovery time correlates

with bond reforming time, Nature, 414(2001) 773.

86 Parbhu AN, Bryson WG & Lal R, Disulfide bonds in the

outer layer of keratin fibers confer higher mechanical

rigidity: Correlative nano-indentation and elasticity

measurement with an AFM, Biochemistry, 38 (1999) 11755.

87 Ramachandran S, Cohen D, Quist AP & Lal R, Imaging and

Optical Microscopy – III, Biophysical Journal, 94 (22008)

280.

88 Ionescu-Zanetti C, Mechler A, Carter SA & Lal R,

Semiconductive polymer blends: Correlating structure with

transport properties at the nanoscale, Advanced Materials,

16 (2004) 579.

89 Jager EWH, Smela E & Inganas O, Microfabricating

conjugated polymer actuators, Science, 290 (2000) 1540.

90 Hagleitner C, Hierlemann A, Lange D, Kummer A, Kerness

N, Brand O & Baltes H, Smart single-chip gas sensor

microsystem, Nature, 414 (2001) 293.


1036

91 Carter SA, Angelopoulos M, Karg S, Brock PJ & Scott JC,

Polymeric anodes for improved polymer light-emitting

diode performance, Appl Physics Lett, 70 (1997) 2067.

92 Quist AP, Chand A, Ramachandran S, Daraio C, Jin S & Lal

R, Atomic force microscopy imaging and electrical

recording of lipid bilayers supported over microfabricated

silicon chip nanopores: Lab-on-a-chip system for lipid

membranes and ion channels, Langmuir, 23(2007) 1375.

93 Seo J, Ionescu-Zanetti C, Diamond J, Lal R & Lee LP,

Integrated multiple patch-clamp array chip via lateral cell

trapping junctions, Appl Physics Lett, 84 (2004) 1973.

94 Xu J, Wang XB, Ensign B, Li M, Wu L, Guia A & Xu JQ,

Ion-channel assay technologies: quo vadis? Drug Disc

Today, 6 (2001) 1278.

95 Andersen JET, Zhang JD, Chi Q, Hansen AG, Nielsen JU,

Friis EP, Ulstrup J, Boisen A & Jensenius H, In situ scanning

probe microscopy and new perspectives in analytical

chemistry, Trac-Trends Analytl Chem, 18 (1999) 665.

96 Boisen A, Thaysen J, Jensenius H & Hansen O,

Environmental sensors based on micromachined cantilevers

with integrated read-out, Ultramicroscopy, 82 (2000) 11.

97 Jensenius H, Thaysen J, Rasmussen AA, Veje LH, Hansen

O & Boisen A, A microcantilever-based alcohol vapor

sensor-application and response model, Appl Physics Lett,

76 (2000) 2615.

98 Hauptmann P, Lucklum R, Puttmer A & Henning B.

Ultrasonic sensors for process monitoring and chemical

analysis: State-of-the-art and trends, Sensors and Actuators

A-Physical, 67 (1998) 32.

99 Mechler A, Piorek B, Lal R & Banerjee S, Nanoscale

velocity-drag force relationship in thin liquid layers

measured by atomic force microscopy, Appl Physics Lett, 85

(2004) 3881.

100 Quist A, Chand A, Ramachandran S, Cohen D & Lal R,

Piezoresistive cantilever based nanoflow and viscosity

sensor for microchannels, Lab on a Chip, 6(2006) 1450.

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