Nanotechnology for Disease Screening and Diagnostics
Manda Williams and Pei-Tzu Wu
Abstract
A major emphasis in bioengineering and medical technology is improved diagnostic
techniques to screen for disease. Such screening is required to identify illnesses,
assess risk of disease onset, or determine progression or improvement of disease
state for diseases such as cancer, stroke, Alzheimer’s, or cardiac disease.
Nanotechnology may improve the sensitivity, selectivity, speed, cost, and
convenience of diagnosis. Individual biomolecular interactions can be detected by
the deflection of a microcantilever, the red-shifted emission of a gold nanoparticle,
or the altered conductance of a nanowire. Nanoscale labeling agents, such as
quantum dots, have numerous advantages to intracellular labeling and
visualization. Nanotechnology has opened up the possibility of other screening
strategies as well. These techniques and others can be further developed to enable
enhanced visualization of an array, cell culture, or tumor; be multiplexed to create
smaller, denser gene and protein chips; or be integrated into a handheld nanofluidic
device to improve clinical diagnosis of disease.
Introduction
Correct and rapid identification and quantification of disease is important to effective
treatment, and is a major emphasis of biotechnological and medical research currently.
New screening techniques and practices for cardiovascular disease seem to be in the news
almost weekly. Challenges 13 and 14 of the Bill and Melinda Gates Foundation’s “Grand
Challenges for Global Health” pertain to rapid screening to assess population health and
to on-site diagnosis of febrile diseases to speed proper treatment1. Screening is used to
detect disease prior to the onset of clinical symptoms, with techniques such as
mammography2; to correctly identify disease in the presence of ambiguous symptoms,
the goal of the Grand Challenge #14; estimate risk prior to disease onset, as by
measurement of serum cholesterol; and to quantify disease to assess changes in disease
state, such as the measurement of prostate specific antigen to determine the progression
of prostate cancer or the efficacy of treatment. Improvement in disease screening could
minimize damage and enable more effective treatment by allowing the correct course of
action to be identified earlier in the disease state, reduce incidence of serious disease by
encouraging targeted preventative measures, and improve management of disease by
providing more detailed information on the patient’s status.
Nanotechnology may enhance disease screening by improving sensitivity, selectivity,
time to diagnosis, and the availability of testing equipment. Often, the differences
between healthy and diseased or pre-disease states are very small, and the ability to detect
single molecules or small changes in the behavior of a cell is required for diagnosis.
Technology capable of measuring single binding events or interactions on the nanoscale
would be a great asset. Because the techniques are so sensitive, errors such as binding
mismatches could be identified as such, reducing the incidence of false positives.
Nanotechnology is capable of producing screening techniques much more accurate than
those currently in use. Furthermore, because many of these techniques measure the
molecules and cells directly, without intermediate reactions and sample processing, the
tests are considerably more rapid, and could potentially be conducted entirely in a
doctor’s office or lab down the hall, rather than being sent out to a lab. This reduces the
time between testing and the initiation of treatment, potentially by several days.
Additionally, the elimination of intermediate handling also reduces the potential for error
due to contamination or mishandling. Furthermore, fewer steps may mean less equipment
is required for the storage and processing of samples, reducing the need for a larger
facility and increasing the feasibility of testing on-site. Nanotechnology has the potential
to dramatically improve disease screening, which would result in a significant
improvement in treatment efficacy and ultimately in prognosis and patient experience.
Detection of Chemical Analytes
 Mechanical Detection
Micro-cantilever sensors utilizing the piezoresistive readout can tell us the cantilever
bending deflection resulted from the surface stress change due to the binding events.1
Piezoresistivity: the electrical resistivity of a piezoresistive film (poly-Si) varies with
applied stress. On the basis, the nano-mechanical biosensor make this technology
promising for high-throughput and label-free protein analysis. By Stoney (1909), the
relationship between surface stress and the deflection can be calculated by the following
formula.
Et 2
 
z
3(1   )l 2
σ: differential surface stress, υ: Poisson ratio, E: the Young’s modulus, t: the thickness of
cantilever, l: the length of cantilever, z: the deflection
Figure 1: Schematic of monoclonal anti-PSA and CRP immobilization using Calixcrown SAMs on
Au surface of piezoresistive micro-cantilever sensor.
Figure 2: Real-time monitoring of the micro-cantilever deflection signal as a function of PSA
concentration
Based on similar deflection basis, micro-cantilevers coupled with position-sensitive diode
detector and laser beam are used as a transducer of the antibody-antigen binding
interaction as shown in Figure 7.12 The deflection of microcantilever originates from free
surface energy change induced by the specific biomolecular binding is optically detected.
Two forms of prostate-specific antigen (PSA) can be detected over a wide range of
concentration in a background of human serum albumin (HSA) and human plasminogen
(HP). The relationship between cantilever deflection and geometry and surface stress is
addressed and studied (Figure 9). Protein signature patterns, reflecting different stages of
the disease, can be readily detected using immunosensor chips based on multiple
antibody-functionalized transducers. The idea of "nanolab" chip is introduced to
microcantilever or nanowire methods that individual cells flow across the device while
nanowire sensors identify genes and proteins and nanomechanical sensors detect protein
and gene interactions. This integrated devices and systems will be discussed later.
Figure 3: Schematic diagram of the experimental setup showing a fluid cell within which a
microcantilever beam was mounted. The scanning electron micrograph on the right shows a gold-
coated silicon nitride cantilever beam that was 200 μm long, 0.5μm thick and with each leg 40μm
wide.12
Figure 4: Detection of complex PSA (cPSA).Cantilever deflection versus time for detection of cPSA in
presence of 1 mg/ml of BSA using 200-μm-long and 0.5-μm-thick silicon nitride microcantilevers.
Figure 5: Steady-state cantilever deflections as a function of fPSA and cPSA concentrations for three
different cantilever geometries. Note that longer cantilevers produce larger deflections for the same
PSA concentration, thereby providing higher sensitivity.
 Electrical Detection
In disease diagnostics, the detection of disease-related proteins (disease markers) is the
major concern under study. Other targets such as DNA hybridization or RNA expression
profiling are also studied to help disease identification and diagnosis. Those molecular
events such as antigen-antibody binding (protein-protein interaction) can be detected by
different readout signals such as piezoresistive response1, conductance change2-6, and
voltammetry7.
In the cases of silicon-based nanowires2,3 and carbon nanotubes4-6 in array formats, when
biomolecules (bound to the surface of carbon nanotubes or nanowires) undergoing a
binding event with conformational change or change of charge state, may perturb the
current flow in the nanotubes and nanowires. The nanowires and carbon nanotubes are
integrated on field effect transistor (FET) devices to facilitate the electrical readout. For
the chemically sensitive FETs, the binding event is to be monitored by a direct change in
conductance or other related electrical property. With applied nanotechnology, electrical
detection is even more attractive because electrical systems can be miniaturized and
integrated into systems and provide sensitive, specific, label-free, and real-time
multiplexed detection in proteomic or genomic analysis.
Figure 6: (a) Optical image (top) of a nanowire device array. The white lines correspond to the
silicon nitride passivated metal electrodes that connect to individual nanowire devices. The red
rectangle highlights one of the repeated (vertical) regions where the nanowire devices are formed (b)
Schematic showing two nanowire devices, 1 and 2, within an array, where the nanowires are
modified with different antibody receptors. A cancer marker protein that binds specifically to its
receptor will produce a conductance change characteristic of the surface charge of the protein only
on nanowire-1. (c) Change in conductance versus concentration of PSA for a p-type silicon nanowire
modified with PSA-Ab1 receptor. Inset: Conductance-versus-time data recorded after alternate
delivery of PSA and pure buffer solutions. (d) Schematic illustrating multiplexed protein detection by
three silicon-nanowire devices in an array. Devices 1, 2 and 3 are fabricated from similar nanowires,
and then differentiated with distinct mAb receptors specific to three different cancer markers.
It has been demonstrated that CdS, ZnS, CuS, and PbS nanoparticles or quantum dots
were used to differentiate the signals of four proteins or DNA targets along with stripping
voltammetry of the corresponding metals.7,8 Each binding event yielded a distinct
voltammetric peak, whose size and position reflected the level and identity, respectively.
Figure 7: Simultaneous monitoring of multiple proteins in connection to different inorganic
nanocrystal tags.8
 Optical Detection and Visualization
Optical detection remains the most widely used mechanism for detecting biological
events and for imaging in biological systems.
Nanoparticles or quantum dots (QDs), used as tags or labels, increase the sensitivity,
speed, and flexibility of detecting or measuring the presence or activity of selected
proteins or DNA. Semiconductor nanoparticles or QDs have been conjugated with
biorecognition molecules such as peptides, antibodies, nucleic acids or ligands for
application as fluorescent probes. The interesting and attractive optical properties such as
size- and composition-tunable fluorescence emission from visible to infrared
wavelengths, and good photostability can enhance their applications as tags or encoding
proteins or DNAs. CdSe quantum dots exhibit photostability that the red core in the upper
panels and the green core in the lower panels in Figure 5(a) compare to the region
surrounding the core. Moreover, the new nanostructure shown in Figure 5(b) may be used
as a biological label with polarized emission, reduced blinking and faster radiative rates
than dots potentially.
Figure 8: (a) Photostability of CdSe QDs vs. the dye Alexa 488 (b) Transmission electron
micrographs of quantum rods-a new nanostructure of quantum rods may use as biological label with
polarized emission.9
Except QDs, gold nanoparticles are also widely studied for biological uses. One of their
interesting applications is to be used as a color reporting group for signaling molecular
recognition events and make the nanomolar concentration detection possible. Upon
different agglomeration states induced by protein-protein interaction on the surface of
gold nanoparticles, there is a distinct color change to naked eyes readily.11
Figure 9: A schematic illustration for the colorimetric detection of protein-protein interaction
utilizing gold nanoparticles.11
In vitro diagnostics has no safety issues of fate of nanoparticles in the human body.
Although in vivo cancer targeting and imaging with semiconductor quantum dots10 has
been studied and demonstrated in mice, the toxicity of QDs to living animals is not
evaluated yet. In the future, the goal will be to enable single molecule detection in vivo,
despite the large background present in a living system.
Alternative Detection Methods
Though detection of chemical analytes and visualization are favored methods of
screening, other possibilities exist, made possible by nanotechnology. The most attractive
at this point is mechanical probing of cells.
Cells in vivo are subject to a variety of mechanical inputs, ranging from the tension on
muscle fibers and skin cells, to the compressive forces exerted on bone and the pulsatile
pressures on endothelium lining the arteries. The mechanical properties of these cells and
their responses to these mechanical stimuli are crucial to their function. For most cells,
the cytoskeleton, a network of proteins, is the primary determinant of shape and
mechanical properties. In addition to moving chromosomes and organelles during cell
division, the cytoskeleton also links the receptors protruding through the membrane,
positioning them, and acts as a highway for vesicle traffic in the cell16. The system is in a
dynamic equilibrium, with a constant assembly and disassembly of microtubules and
remodeling to allow the cell to respond to new stimuli, such as the clustering of receptors
initiated by adhesion to a surface. Largely due to its role in cell division, the cytoskeleton
has been a major target for anti-cancer therapeutics, but the rapid replication and growth
also result in tumor cells having a different morphology and mechanical properties.
Figure 10: The longer the delay between an applied torque and a shear, the more uniform a cell's
response, due to remodeling of the cytoskeleton. 15
Ultrasound has been utilized to detect cancer, but the contrast is relatively low in many
cases, so a tumor in dense tissue may be missed17. The few current methods that utilize a
mechanical screen are dependent upon bulk properties of the tissue, and do not have
sufficient resolution to detect low numbers of cancerous or pre-cancerous cells. However,
ultrasound has been used to measure the reflection coefficients, elasticity, spectral
responses, and internodal distance of normal and malignant breast tissue on the nanomechanical scale18. This rethinking of traditional ultrasound analysis could increase its
utility in cancer screening.
The cell is not a static system, and the dynamics of cytoskeletal remodeling would be
altered in a cancerous or precancerous cell. Some consider the cell and cytoskeleton to be
a soft glassy system; in response to stress, the cytoskeleton undergoes small jumps to
increasingly stable configurations15. The system may be rejuvenated by applied shear. In
the abnormal environment of a cancerous or pre-cancerous cell, however, these processes
would have different parameters. Though this method would require a tissue sample,
shear measurements and sample visualization could be achieved by atomic force
microscopy. By combining indentation data with imaging, AFM can reveal the spatial
distribution of viscoelastic properties in the cell, effectively showing the structure of the
cytoskeleton19. While imaging single cells would be tedious, perhaps the sensitivity of the
technique could be developed to answer the question of whether or not the patient has or
is at risk for cancer with a relatively small sample.
Figure 11: Normal, and increasingly aberrant behavior in human breast duct cells,
http://www.uphs.upenn.edu/news/News_Releases/sep05/tissuetumform.htm
Not all research targets made available by nanotechnology, however, are suitable for
diagnostics. For example, electron transfer by proteins is crucial to many important
biological processes such as DNA repair activation and cellular respiration, and scanning
tunneling microscopy and electrochemical measurements can be used to detect and
quantify these currents20. However, though such research will enhance understanding of
basic cellular and metabolic mechanisms and pathologies, disorders of these systems are
not well understood, and diagnosis in a patient would effectively be worthless at this
point. Identifying disease is only clinically meaningful if a possibility of treatment exists.
The ideal diagnostic targets, therefore, are not novel, but rather well-studied properties
with known roles in disease, for which nanotechnology enables a practical method of
screening.
Integrated Devices and Systems
In order for a screening technique to be clinically useful, it must give a clear and relevant
answer, with a minimum of complication to the testing protocol. Currently, many
methods in practice give information on only a single parameter, which may not even be
an ideal target for screening, and may be inaccurate due to handling and other introduced
error. Furthermore, if the analysis lab is not on-site, the sample must be sent out, resulting
in a delay that can be anywhere from several hours to over a week, and the additional
potential problems of storage and contamination. The possibility of simple, point-of-care
diagnostics to reduce inaccuracy and wait time is a major motivation for the field of
microfluidics. Such “lab-on-a-chip” technology incorporates functionality such as PCR
and ELISA units into handheld devices, and will dramatically improve healthcare,
particularly in the developing world1.
Nanotechnology should have many of the same advantages as the chemistry used in
microfluidic devices. The small focus and direct data acquisition of nanotechnological
methods are an asset, allowing more information to be obtained from a sample with
minimal processing and equipment. This enables multiple analytical techniques to be
incorporated into a single device or system. Nanowires and microcantilevers could easily
be utilized in a microfluidic or nanofluidic device21. A microfluidic flow cell built on a
gold film can be used for monitoring of the surface and binding by SPR. The addition of
nanotechnology chemical analysis techniques to microfluidic devices is not a large leap
conceptually or technologically.
Due to the wide choice of spectra for nanoparticles and quantum dots, multiple targets in
the cell may be visualized. Additionally, they can be visualized using a single excitation
source, minimizing damage to the cells. The ability of nanoparticles to enter the cell
without harming it open up the possibility of examining the morphology, chemistry, and
presence of one or more targets in a cell simultaneously with labeling probes and
PEBBLEs (Probes Encapsulated By Biologically Localized Embedding). Furthermore,
the responses of the cells to chemical or mechanical stimuli could be assessed and
visualized in the cultured sample.
Figure 12: FIRAT simultaneously collects a variety of information: (from upper left to right)
topography, adhesion energy, contact time and stiffness.22
Non-optical screening of cells does not seem at first thought to be practical in a clinical
setting. A distribution of cell properties would be preferable for early diagnosis of cancer
to bulk tissue properties, and this requires screening of many cells in a sample. One AFM
method that may revolutionize mechanical screening of cells is FIRAT: Force Sensing
Integrated Readout and Active Tip22. The tip used is smaller, enabling collection of
detailed topographical, adhesive, and mechanical information as fast as 60Hz—so movies
of dynamics can be produced. Furthermore, modulated vibration of these tips could
collect frequency spectra and internodal distances rapidly enough to yield such data for a
reasonably large sample of cells in a reasonable amount of time. An array of AFM
cantilevers would vastly increase the speed and range of data acquisition from a cell
culture. Additionally, multiple techniques may be used simultaneously: along with
topographical and stiffness measurements, adhesion to a tip modified with a substrate
molecule could assess receptor distribution, or presence of a particular target compound
in the culture could be measured by binding to nanowires.
References
1.
2.
“Grand Challenges in Global Health, Grand Challenge #14.” 2005.
http://www.grandchallengesgh.org/ArDisplay.aspx?SecID=344&ID=64
Soper, SA. “Center for Biomodular Multi-Scale Systems: Engineering.” Louisiana
State
University,
2005.
http://appl003.lsu.edu/cbmm/cbmm.nsf/$Content/Engineering?OpenDocument
3. Wee KW, Kang GY, Park J, Kang JY, Yoon DS, Park JH, Kim TS. “Novel electrical
detection of label-free diseasr marker proteins using piezoresistive self-sensing
micro-cantilevers.” Biosensors and Bioelectronics. 2005. 20, 1932-1938.
4. Cui Y, Wei Q, Park H, Leiber CM. “Nanowire nanosensors for highly sensitive and
selective detection of biological and chemical species.” Science. 2001. 293, 12891292.
5. Zheng G, Patolsky F, Cui Y, Wang WU, Leiber CM. “Multiplexed electrical
detection of cancer markers with nanowire sensor arrays.” Nature Biotechnology.
2005. 23(10), 1294-1301.
6. Chen RJ, Bangsaruntip S, Drouvalakis KA, Kam NWS, Shim M, Li Y, Kim W, Utz P
J, Dai H. “Nanocovalent functionalization of carbon nanotubes for highly specific
electronic biosensors.” Proc. Natl. Acad. Sci. U.S.A. 2003. 100, 4984-4898.
7. Chen RJ, Choi HC, Bangsaruntip S, Yenilmez E, Tang X, Wang Q, Chang Y-L, Dai
H. “An investigation of the mechanisms of electronic sensing of protein adsorption on
carbon nanotube devices.” J. Am. Chem. Soc. 2004. 126, 1563-1568.
8. Staii C, Johnson Jr. AT. “DNA-decorated carbon nanotubes for chemical sensing.”
Nano Lett. 2005. 5(9), 1774-1778.
9. Wang J. “Electrochemical biosensors: Towards point-of-care cancer diagnostics.”
Biosensors and Bioelectronics. 2005. in press.
10. Liu G, Wang J, Kim J, Jan MR. “Electrochemical coding for multiplexed
immunoassays of proteins” Anal. Chem. 2004. 76, 7126-7130.
11. Alivisatos P. “The use of nanocrystals in biological detection.” Nature Biotechnology.
2004. 22(1), 47-52.
12. Gao X, Cui Y, Levenson RM, Chung LWK, Nie S. “In vivo cancer targeting and
imaging with semiconductor quantum dots.” Nature Biotechnology. 2004. 22(8), 969976.
13. Tsai C-S, Yu T-B, Chen C-T. “Gold nanoparticle-based competitive colorimetric
assay for detection of protein-protein interactions.” ChemComm. 2005. 4273-4275.
14. Wu G, Datar RH, Hansen KM, Thundat T, Cote RJ, Majumdar . “Bioassay of
prostate-specific antigen (PSA) using microcantilevers.” Nature Biotechnology. 2001.
19, 856-860.
15. Bursac P, Lenormand G, Fabry B, Oliver M, Weitz DA, Viasnoff V, Butler JP,
Fredburg JJ. “Cytoskeletal remodelling and slow dynamics in the living cell.” Nature
Materials.
2005.
4(7),
557-561.
http://www.nature.com/nmat/journal/v4/n7/pdf/nmat1404.pdf
16. Alberts, Johnson, Lewis, Raff, Roberts, Walter. Molecular Biology of the Cell, 4th ed.
2002.
17. Tilanus-Linthorst M, Obdeijn I, Bartels K, de Koning H, Oudkerk M. “First
experiences in screening women at high risk for breast cancer with MR imaging”
Breast Cancer Research and Treatment. 2000, 63(1). 53-61.
18. Liu J, Ferrari M “Mechanical spectral signatures of malignant disease? A smallsample, comparative study of continuum Vs. nano-biomechanical data analyses”
Disease Markers 2002, 18(4): 175-83.
19. Costa KD. “Single-cell elastography: Probing for disease with the atomic force
microscope.” Disease Markers, 2004. 19(2-3) 132-154.
20. Chi Q, Farver O, and Ulstrup J. “Long range protein electron transfer observed at the
single molecule level: In-situ mapping of redox-gated tunneling resonance.” PNAS,
2005. 102(45), 16203-16208. http://www.pnas.org/cgi/reprint/102/45/16203.pdf
21. Zandonella, “Cell nanotechnology: the tiny toolkit” Nature, 2003. 423, 10 – 12.
http://www.nature.com/cgitaf/DynaPage.taf?file=/nature/journal/v423/n6935/full/423010a_r.html&filetype=&d
ynoptions=
22. McRainey M. “New Device Revolutionizes Nano Imaging: Much faster technology
allows AFM to capture nano movies, create material properties images.” (Georgia
Tech
press
release,
Feb.
9,
2006)
http://www.gatech.edu/newsroom/release.php?id=858