Sensors and Actuators B 165 (2012) 16

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Sensors and Actuators B: Chemical

journal homepage: www.elsevier.com/locate/snb

A nanostructured SAW chip-based biosensor detecting cancer cells

Patrick Brker a, , Klaus Lcke b , Markus Perpeet c , Thomas M.A. Gronewold c
a
University of Potsdam, Karl-Liebknecht-Strae 25, D-14476 Potsdam-Golm, Germany
b
GILUPI GmbH, Am Mhlenberg 11, D-14476 Potsdam-Golm, Germany
c
SAW-Instruments GmbH, Schwertbergerstr. 16, D-53177 Bonn, Germany

a r t i c l e i n f o a b s t r a c t

Article history: A nanostructured chip surface was fabricated enabling binding via spaced antibodies specically targeting
Received 3 May 2011 surface proteins of cancer cells and detection of extremely low numbers of circulating tumor cells (CTC)
Received in revised form 2 November 2011 without labeling using a sam 5 biosensor. The antibody surfaces mostly were generated by self assembly
Accepted 8 November 2011
of antibodies to gold nanospots on the sensitive SiO2 -surface of a sam 5 chip. Compared with a complete
Available online 16 February 2012
gold surface, only 40% of the amount of antibodies was bound to the nanospot surface, but structured
such that 15-fold higher sensitivity to vital cancer cells was achieved. Human cancer cell lines JEG-3
Keywords:
(lymphoblastic leukemia) and MOLT-17 (placental choriocarcinoma) from cell cultures were successfully
Nanospots
Nanostructured surfaces
detected. The sensor showed signicant responses on less than 10 cells injected in a single run. The
SAW sensor extreme increase in sensitivity and its simple regeneration emphasizes the usefulness of its introduction
Cell detection in biomedical applications.
Cancer cells 2011 Elsevier B.V. All rights reserved.
sam 5

1. Introduction needs to be produced. For the presented nanostructured surfaces,

Application of nanotechnology shrinks feature sizes. Nanopar-
ticles have a large surface-to-volume ratio compared to bulk [1].
This changes physical and absorption properties of nanoparticles
in solution [2]. Electrochemical applications like electron tunneling
are enabled. Surface modications change the chemical properties
for binding reactions [1]. In many biosensors, biological material
is applied to nanoparticles or the nanostructures on a nonbinding
surface. In the majority, gold nanoparticles are fabricated. Nanos-
tructures [35] are applied (1) in medicine [6,7], (2) for diagnostics
[8] as in lab-on-a-chip applications, nanoparticles, or as quantum
spots [9,10], (3) for drug delivery [11] and implantable delivery sys-
tems to reduce doses [12] and for transportation to the targeted site
of action [13], or (4) to enhance catalysis [14].
Despite the advantages indicated, surfaces modied with nanos-
tructures still are researched to a lesser extent because of their
sophisticated and challenging fabrication. Only little variance can
be tolerated. Nanoparticles have been caged on surfaces to con-
strain nanospots [15,16], or they have been prepared by clustered
sputtering [17]. To repeatedly achieve comparable results, exactly
the same pattern of dened nanostructured islands on a surface
Abbreviations: CTC, circulating tumor cells; IgG, Immunoglobulin G; SAM, self
assembled monolayer.
Corresponding author.
E-mail address: Patrick.broeker@gmx.de (P. Brker).
small regions of one material (gold) are applied to a basis com-
posed of another material (SiO2 ). Self-assembled spots typically
range from 100 to 1000 nm. By use of a structured lithographic
mask, the number, size and spatial resolution of spots is precisely
dened with lateral dimensions not exceeding 100 nm.
Mostly, nanoparticles and nanostructures are modied by
established protocols and traditional surface chemistries. The
experiments in this article were performed on small sized gold
islands, to which untagged antibodies were adsorbed. Self assembly
of ligands to a nanospot surface is an unmatched simple and fast
method, often providing highly efcient surfaces. Also, toxic side
effects are prevented on such modied nanoparticles and nano-
materials intended for medical applications [18]. However, the
molecules are attached randomly. Antibodies also were bound via
an interface of self assembled monolayer (SAM). A surface was
created bearing carboxylic end groups enabling subsequent cou-
pling chemistries [19]. Oriented ligands properly presenting their
analyte-binding sites are only obtainable by specic binding. On
two-dimensional surfaces, the density of ligands also is of great
importance. By tight packing of antibodies for example, binding
of the respective antigen is sterically hindered. Such spatial inter-
ference is counteracted by the introduction of articial borders
of non-binding materials surrounding islands of binding surfaces.
The size of such islands needs to be optimized for binding of small
numbers of antibodies [20]. SiO2 microposts were fabricated on a
surface with a height and diameter of 100 m, spaced by an average
gap of 50 m. Various tumor cells circulating in the blood of cancer

0925-4005/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.snb.2011.11.022
2 P. Brker et al. / Sensors and Actuators B 165 (2012) 16
patients were bound at 51281 cells/ml. For optimal cell binding,
a lower limit of 40 bronectin molecules per binding nanoparticle
was dened by Pesen and Haviland [21].
The development of hybrid surfaces based on universal
platforms at the bioticantibiotic interface [22] enables new appli-
cations or lowers the detection limit. Current methods for detection
of very low numbers or even single CTCs are either direct imag-
ing, or immunocytochemical and molecular assays [23]. Methods
like the CellSearchTM Assay (Nichols Institute) detect CTCs using
antibody-based immunomagnetic cell enrichment enhanced by
nucleus labeling with uorescent dye. According to the manufac-
turers instruction the detection limit is 1 CTC/7.5 ml blood (<5 for
metastatic breast cancer and prostate cancer, and <3 for metastatic
colorectal cancer). Many such highly efcient devices are destined
for one-time use only and need several processing steps, which
extends the production cost and time as well as the duration of one
assay [24].
The technological basis for a nanospot-based biosensor with
increased sensitivity for proteins and cells was a surface acous-
tic wave (SAW) sam 5 blue sensor [25,26]. The sensor measures
phase and amplitude signal, which is related to mass and viscosity
alteration. On sam 5 chips with a sensitive SiO2 surface [27,28],
spatially conned gold nanospots were fabricated. Antibodies
preferably bind to the gold surfaces, but not to the surround-
ing SiO2 material. By this method, antibodies specically binding
cell surface proteins were immobilized on the nanospots. The
resulting surface displayed patches of few, clustered antibodies,
optimized to detect small numbers of cells. The created sensor
was applied to medically relevant systems. Human cancer cell lines
were selectively bound to the sensor: (1) MOLT-17, from a T-cell
lymphoblastic leukemia cell line, (2) JEG-3, a choriocarcinoma cell
line, and (3) osteoblasts. The sam 5 shows minimal sensitivity to
pH and buffer changes and, thus, it is predestined for the analysis
applied. The kinetics and efciency of cell binding to the nanostruc-
tured surfaces was investigated and compared with complete gold
surfaces.
2. Experimental
2.1. Materials
All chemicals were of analytical grade and used as received,
except stated otherwise. Cell binding buffer was PBS, pH 7.4
of 14 mM NaCl, 0.3 mM KCl, 1 mM Na2 HPO4 . The buffer sub-
stances were obtained from Sigma. Cells were diluted in binding
buffer. Anti-HLA-G (cloneMEM-G9) was bought from Exbio and
anti-CD4 antibodies from Chemicon (CBL131). As a reference, a
random mixture immunglobulin G (I-2511) was purchased from
Sigma.
2.2. SAW sensor chips
Love-wave sensor chips with ve sensor elements and a sen-
sitive area of SiO2 were prepared from ST-cut quartz substrates.
A Cr/Au/Cr layer was structured by standard photolithography
to shape interdigital transducers and contact pads. Details are
described by Schlensog et al. and Perpeet et al. [29,30]. A SiO2
guiding layer was then deposited not overlapping the IDTs,
functioning as both electric shielding and surface as an inter-
face to biological experiments. Finally, the wafer was cut into
20 mm 14 mm sensor chips, each carrying ve independent sen-
sor structures of 1.7 mm 4 mm. The SiO2 surface was further
processed with the nanostructures by using wet-lithographic
methods [31,32]. We designed a single layer of latex parti-
cles of appointed size by self assembly on water which is
described in detail in Patent 20090131274 [32]. Oriented latex
particles were transferred to the SiO2 surface of sam 5 chips
to be used as a photolithographic mask. Gold was evaporated
into the resulting gap between the ordered particles, generat-
ing nanoparticles of dened interspace and size (Fig. 1). The
size of nanoparticles depends on packing properties. The sur-
face surrounding one particle can be calculated as the surface
of half a parallelogram with Aparticle = D2 cos 30 , resulting
in 83,831 nm2 particle1 at D = 440 nm. The number of nanospots
can then be calculated for a given surface area of 1 mm2
with 1 mm2 /8.38 108 = 1.193 107 particles mm2 , correspond-
ing approximately 1.2 109 particles cm2 .
For the experiments, acoustic waves were excited perpendicu-
lar to the crystallographic X-axes at a working frequency between
147 and 150 MHz independent of the electroacoustic resonance.
We focus in this article on an evaluation of the phase signal element.
The sam 5 blue sensor used records both phase and amplitude sig-
nal, separately reecting mass changes and changes in viscoelastic
properties, respectively. The recorded mass changes represented in
the phase signal are essentially not interfered by the buffer com-
ponents. Measurements were recorded on-line in real time. The
concentration was calculated using the sensitivity of 515 cm2 /g
[29].
2.3. Modication of the sensor chip surfaces and measurement in
the microuidic SAW sensor system
A SAM of 11-mercaptoundecanoic acid (Sigma) was generated
externally on the gold fraction of the shielding [29]. Further bind-
ing steps were performed in the biosensor. The sensor chips were
placed into a sam 5 read out system. Mass loading and viscoelas-
tic alterations of biomolecular interaction processes on the surface
of the sensor chip are recorded in real time as signals changes
in phase shift and amplitude, using a standard PC [29,30]. This
enables precise detection and separation of mass from viscos-
ity alterations. Surface modications and the binding cycles were
recorded. The samples were injected by means of an autosampler
into a constant buffer stream at a ow rate 30 l/min and con-
stant temperature of 23 C. The carboxyl groups were activated at
constant H2 O ow using 200 mM N-(3-dimethylaminopropyl)-N-
ethylcarbodiimide (EDC) and 50 mM N-hydroxysuccinimide (NHS).
Antibodies were coupled to the activated SAM using carbodiimide
chemistry [29]. Remaining unreacted NHS-esters were capped
using 1 M ethanolamine, pH 8.0. For cell binding, the running buffer
was replaced with PBS, pH 7.4. Cells were diluted in running buffer
and injected into the buffer stream.
2.4. Cancer cellantibody systems
Cancer cells used are from human T-cell line (MOLT-17, T-cell
line; Thrombocyte-cancer hybrid cells, T-cell acute lymphoblastic
leukemias (German Collection of Microorganisms and Cell Cultures,
DSMZ ACC 36)). These cells express on the surface CD4-antigen and
are non-adherent. For binding of the cells, anti-CD4 antibody was
used.
Human JEG-3-cells are from a choriocarcinoma (ECACC
92120308, American Type Culture Collection ATCC HTB 36; derived
from DSMZ). They express and expose HLA-G on the surface. For
binding experiments, the anti-HLA-G antibody (Clone: MEM/G9,
Batch-No. 070207) and the anti-HLA-G-F(ab) were purchased from
AbD SEROTEC, AbD05681.1.
Osteoblast-like cells were used to control selectivity of binding
the CD4 cells. These cells were described from Giannona et al. [33].
 P. Brker et al. / Sensors and Actuators B 165 (2012) 16 3

Fig. 1. AFM (left) and SEM image (right) of the chip surface with nanospots sputtered on an SiO2 chip surface. The drawn nanoparticles indicate their size and packing
properties. The surface surrounding one particle can be calculated as the surface of half a parallelogram with Aparticle = D2 cos 30 , resulting in 83,831 nm2 particle1
at D = 440 nm. The number of nanospots can then be calculated for a given surface area of 1 mm2 with 1 mm2 /8.38 108 = 1.193 107 particles mm2 , corresponding
approximately 1.2 109 particles cm2 .

3. Results expanding the experiment to binding with and without a SAM

3.1. Selecting size and spacing gap between nanospots
Nanostructured sam 5 chips modied with selective
biomolecules are introduced to increase sensitivity for vital
cells and other antigens without labeling. Size and shape of the
applied nanospots is determined by both diameter and pattern of
the particles used.
Originally tested were polystyrene beads with a diameter from
200 to 2000 nm [31]. Gold was sputtered in shadow nanosphere
lithography [31,33]. Periodic and highly regularly arranged metallic
two-dimensional nanostructures were produced, using the ordered
beads as the mask dictating the shape of the resulting nanoparti-
cles [35]. We nally selected polystyrene beads with a diameter of
D = 440 nm in hexagonal arrangement on SiO2 surfaces of sam 5
chips. The properties of the mask used resulted in nanoparticles
with roughly triangular shape as conrmed by AFM and SEM char-
acterization (Fig. 1). For highly specic and selective binding, the
nanospots were modied with antibodies. On the resulting chips,
the amount of nanoclustered antibodies was optimized to maxi-
mize cell binding.
interface, data were conrmed for the nanospot surface with
Nanospot (5) = 2.9 , while the complete gold surface showed a con-
siderably lower binding with Au (5) = 7.9 (Table 1). In all sets of
experiments, the nanospot surface was proven to show very reli-
able binding with relatively little variations from the mean and few
outliers.
The recorded signals were related to mass loading using the sen-
sitivity factor to compare the binding capacity (Fig. 2; Table 1). The
total amount of adsorbed antibody was about 2.4-times higher on
the plain gold surface than on the nanostructured surface. The use of
an orienting SAM increased the signal of antibodies binding to both
surfaces by 27% on the nanostructured surface, and by 15% on the
plain gold surface, increasing the binding factor to >2.6. In average,
about cnanospot (5) = 5.6 ng/cm2 antibody was bound to the nanos-
tructured chip surface, and cAu (5) = 15.2 ng/cm2 was bound to the
gold covered surface (average of ve). This supports the assump-
tion that the self assembled antibodies conned to the nanospots
were not randomly bound, but clustered in xed spacing.
3.3. Calculation of number of antibodies per nanospot and of cells
on the chip surface

3.2. Amount of antibodies bound to nanostructured and complete
gold surfaces
Antibodies were coupled to sam 5 chips with a nanostruc-
tured or a complete gold surface. Concluding from a range of
measurements, the nanospot surface is saturated at injection of
500 ng/ml anti-CD4 antibody with  2.8 . Binding of anti-
CD4 antibody without an interface resulted in Au (average of
20) = 10.1 22.5% and Nanospot (25) = 2.8 27%. In another set
The nanostructured surface capturing MOLT-17 cells (Fig. 3)
typically showed a phase shift from anti-CD4 antibody in
the range of 3 . With the sensitivity factor and an assumed
molecular weight of the antibody of 150,000 equals about
38.3 fmol/cm2 = 3.8 1013 M. With the Avogadro constant of
NA = 6.022 1023 mol1 (molecules per mol), the number of bound
antibodies is calculated with 2.3 1010 antibodies/cm2 . The num-
ber of nanospots is about 1.193 109 particles cm2 (Fig. 1),
equalling to in average about 19.3 antibodies per nanospot. This

Table 1
Evaluation of phase shifts of antibody binding from sam 5 chips with nanostructures and with a gold surface, and with or without a SAM of 11-mercaptoundecanoic acid.
The coverage was evaluated using the sensitivity factor and an assumed antibody size of 150 kDa.

Surface Phase  [ ] Coverage

[ng/cm2 ] [fmol/cm2 ]

Nanostructures Without SAM 2.9 0.8 5.6 1.5 37

With SAM 3.8 0.4 7.5 0.8 50

Gold Without SAM 7.9 3.5 15.2 6.9 101

With SAM 9.1 3.2 17.6 6.2 117
4 P. Brker et al. / Sensors and Actuators B 165 (2012) 16
Fig. 2. Coupling of anti-CD4 antibodies to a nanostructured sam 5 chip surface by adsorption (right) and using carbodiimide chemistry (left) to gold nanostructures (black
line) and to a complete gold surface (red line). Coupling by carbodiimide chemistry to a SAM of mercaptoundecanoic acid resulted in higher antibody density on both
nanostructured and gold covered surface compared to physisorption. (For interpretation of the references to color in this gure legend, the reader is referred to the web
version of the article.)
agrees well with the presumed density based on an estimated size
of an IgG antibody of about 12 nm.
Pesen and Haviland determined in their study the lower limit
for adhesion of cells to surface-presented molecules with about
40 clustered bronectin molecules contained on nanospots with
a radius of about 100 nm [21]. They specify that this number is
required to induce a focal adhesion. Our detected number of 19.3
antibodies per nanospot, each with two binding arms, would be in
excellent accordance with this nding.
The diameter of human cells usually ranges from 10
to 100 nm. JEG-3 and MOLT-17 cells have a size of about
20 m [34]. Assuming a round shape, the theoretical maxi-
mal packing of suspension cells can be calculated according to
Fig. 1 with: the size of a cell with 200,002 cos 30 with
174,000,000 = 1.74 108 nm2 cell1 = 1.7 104 mm2 cell1 . Cells
can be packed to 5747 cells mm2 = 5.7 105 cells cm2 . This
means that roughly >2000 times more antibodies can be bound
to a 2D sensor surface than antibodies.
Fig. 3. Comparison of MOLT-17 cells binding to sensor chips modied with
gold nanostructures (black line) and to a complete gold surface (red line).
Average of the phase shift detected on ve sensor elements with a Standard
Deviation <10%. The anti-CD4 antibody injection resulted in a phase shift of
7.1 13.7 ng/cm2 91.5 fmol/cm2 (complete gold, calculated with the sensitivity
factor) and 3.0 5.7 ng/cm2 38.3 fmol/cm2 (nanospots) or 42%. The rst injection
of 20,000 MOLT-17 cell resulted in a phase shift of 14.14 1.40 (nanospots) and of
0.87 0.82 (complete gold). The total phase change after the injection of 1.8 108
cells was 16.72 1.49 (nanospots) and 17.90 0.25 (complete gold), correspond-
ing to complete coverage. All measurements are independent of each other. (For
interpretation of the references to color in this gure legend, the reader is referred
to the web version of the article.)
3.4. Binding of human leukemic T-cells (MOLT-17)
We decided on using detached MOLT-17 cells to be bound to the
nanostructured chip surface to proof that the antibodies were (1)
presented in bioactive form on the nanospots, (2) in the required
density and (3) in a size and spacing qualied for interacting with
the epitope space at the cell surface. MOLT-17 cells display the
antigen to the highly afne, specic and selective antibody in a
sufciently high concentration on the cell surface.
For the experiments, the cells were freshly diluted in running
buffer PBS, pH7.4 to 2 104 , 2 105 and 1.6 106 cells/ml. 180 l of
those cell suspensions were injected. The mass increase by binding
cells via the antibody was monitored in real-time (Fig. 3).
The results show that the nanostructured surface is highly
sensitive for the cells, especially when compared with the com-
plete gold surface. Even injections of low concentrations of cells
c = 2 104 cells/ml had a relatively high signal, while the gold sur-
face barely gave a detectable signal. Subsequent injections of higher
cell numbers gave little additional signal on the nanostructured
sam 5 chip.
The gold surface was less sensitive. At c = 1.6 106 cells/ml, the
same signal was reached measured on the nanostructures at 80-
times lower concentration. But a comparison of the on-rates shows
that at low cell numbers, the capacity of the nanostructured surface
has been reached, while the gold surface has a much higher capacity
than the tested c = 1.6 106 cells/ml.
3.5. Regeneration
The antibody surface was regenerated using 1 M guanidinium
hydrochloride pH 9.0 and sometimes with 10 mM Glycine, pH 2.2. In
one example, repeated injections of 2 104 MOLT-17 cells resulted
in phase shifts of  = 8.4 0.54 . Typically, the variance in binding
to such surfaces was below 6.5% and often below 5%, an excel-
lent result for such a biological system. After regeneration, the
differences between individual sensor elements eventually were
increased. By its nature, a random fraction of antibodies cannot be
regenerated or is degraded. Also, differences were exaggerated at
very low cell concentrations, because single binding events have a
large impact on the outcome.
3.6. Sensitivity and limit of detection
Cancer cells mostly circulate at low numbers in body uids such
as blood, lymph, or amniotic liquid. Thus, the sensitivity and afn-
ity of the chip has to be increased for binding of low numbers of
 P. Brker et al. / Sensors and Actuators B 165 (2012) 16
Fig. 4. Human JEG-3 cells (black line) and osteoblast cells (red line) binding to a
nanostructured sensor chip modied with anti-CD4 antibody, selectively binding
JEG-3 cells. Average of the phase shift detected on ve sensor elements with a Stan-
dard Deviation <5%. The anti-CD4 antibody injection resulted in a phase shift of
1.3 16.8 fmol/cm2 (calculated with the sensitivity factor). The injections of 50,
500 and 5000 cells/ml resulted in additional phase shifts of 0.22 , 1.74 and 11.47
(JEG-3) and of 0.065 , 0.101 and 0.913 (osteoblasts). (For interpretation of the ref-
erences to color in this gure legend, the reader is referred to the web version of the
article.)
cells. The effectiveness of the nanostructured surface was tested
in a set of experiments with the human placental choriocarcinoma
cell line JEG-3. A nanostructured sam 5 chip surface was modied
with anti-CD4 antibody, recognizing a 59 kDa cell surface glyco-
protein expressed by JEG-3 cells, but not by osteoblast cells which
were used to test the selectivity of the modied surface. Injections
were performed for dilutions of 5000, 500 and 50 cells/ml (Fig. 4).
At an injection volume of 150 l, only 78 cells are injected at
c = 50 cells/ml.
The size of each of the ve elements of the used sensor chips is
7.2 mm2 , which can maximally hold more than 40,000 cells. This
number is much higher than the 101000 cells typically injected
and detected in a single run. The binding of cells then depends on
the afnity of the antibody. A surface covered with high afnity
binders is much faster saturated with cells than a surface with low-
afnity binders. As our experiments show, is this much supported
by its presentation and spacing.
3.7. Selectivity
For use in medical diagnostics, the nanostructured sensor sur-
face is required to bind selected cells with high sensitivity. Two
anti-HLA-G antibodies binding tumor cells were compared with a
third, non-binding surface used as a reference. HLA-G is expressed
by tumors, with which it contributes to the evasion of immuno-
surveillance. Binding to the complete anti-HLA-G antibody clone
MEM/G9 and to a F(ab) fragment thereof (Fig. 5) was compared with
a surface modied with a random IgG mixture. The short F(ab) frag-
ment consists of one constant and one variable domain from each
heavy and light chain of the antibody. At low cell numbers, no sig-
nicant difference was measured between antibody and the F(ab)
fragment. The large antibodies protruding from the surface show a
43% higher binding at c = 5000 cells/ml, based on their higher exi-
bility compared to the globular F(ab) fragments closely attached to
the surface. No or very little binding was measured on the reference
surfaces.
4. Discussion and outlook
A nanostructured sam 5 chip surface was developed and
fabricated (Fig. 1), dening the interspacing between tumor cell
5
Fig. 5. Different numbers of human JEG-3-cells bound by a nanostructured surface
with anti-HLA-G antibody (black line) or an anti-HLA-G-F(ab) fragment (red line).
The reference surface was covered with a random mixture of human immunglob-
ulins. Average of the phase shift detected on ve sensor elements with a Standard
Deviation <5%. The injections of 50, 500 and 5000 cells/ml resulted in additional
phase shifts of 0.18 , 1.69 and 11.47 (MEM-G9), of 0.15 , 1.84 and 6.53 (F(ab))
and of 0.05 , 0.14 and 0.07 (control(blue line)). (For interpretation of the refer-
ences to color in this gure legend, the reader is referred to the web version of the
article.)
surface-specic antibodies. The system is qualied for high-
capacity binding of mobile cancer cells. JEG-3 and MOLT-17 cells
were successfully detected at extremely high sensitivity. The sen-
sor showed signicant response to less than 10 cells injected in a
single run. Compared with a complete gold surface, sensitivity was
increased by more than 100-fold at unchanged high selectivity.
Comparative experiments of antibodies binding by self assem-
bly to the nanostructured and complete gold chip surface were
performed, indicating that only about 40% of the antibodies were
bound to the nanostructures. From measured data, an average of
19 antibodies per nanospot was calculated. This number agrees
well with the number of binders per nanospot optimal for cell
binding [21], arguing for positive cooperativity by multivalent
interactions being advantageous compared with a complete anti-
body surface, or a surface with singled antibodies not strong enough
to capture the bypassing CTCs.
The measurements indicate that the nanostructured sam 5
chips are a highly promising tool for medical diagnostics. Spread-
ing, circulating tumor cells were successfully detected in low
amounts. Without any time-consuming labeling or processing we
were achieving similar sensitivities in the single cell range as are
stated for the most sensitive current methods. But this highly robust
nanostructured chip has an easily regeneratable surface. Successive
detection of cells gave highly comparable results. This achieves high
condence in the measurement. By complete stripping, the chips
are ready for immobilization of binders targeting another set of
CTCs. First experiments with highly sensitive antibodies have been
performed. Simply by use of another set of binders or even engi-
neering of membrane biomimetics [22] the nanostructured sensor
chips can be applied to a completely different set of CTCs. First
experiments detecting proteins and cells from blood, serum and
other unpuried samples with complicated matrices have success-
fully been performed. Details on those experiments, some needing
variation of the interfaces, will be published independently.
In this set of experiments, antibody binding was based on self
assembly to the nanostructures, sufcient to achieve a simple pro-
cedure avoiding toxic components from entering the surrounding
uid. Possibly, an orientation of the antibodies might result in even
higher cell binding, or application of antibodies particularly suitable
or adapted for the nanostructured chip surface. A further increase
might also be achieved by matching sensitive antibodies or other
binders to the special requirements of the nanospot surface.
6 P. Brker et al. / Sensors and Actuators B 165 (2012) 16
Acknowledgments
The authors thank Ulrich Gtz for the chip preparation, cal-
culations and helpful discussions, and Julia Feldner and Melanie
Jungblut for assistance and discussion.
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Biographies
Dr. Patrick Brker studied Biochemistry at University of Hanover, Germany, and
completed his doctorate in 2006 for his work at the institute for Organic Chemistry
(Hanover). Then he changed to the startup company GILUPI GmbH (Bonn; Germany).
After two years he changes to the University of Potsdam in postdoctoral position
(Germany).
Klaus Lcke is now managing director of the company GILUPI GmbH.
Dr. Markus Perpeet studied physics and received his Dr. rer. nat. at the University
of Wuppertal. Additionally, he acquired his MBA at the University of Wales. He
spent several years at different institutes and companies with focus on research
and technology development. He is now one of two Managing Directors of SAW
Instruments (Bonn, Germany).
Dr. Thomas Gronewold studied biology at the Technical University in Braun-
schweig, Germany, and completed his doctorate in 1996 for his work at the Institute
for Biotechnological Sciences (Braunschweig). After a postdoctorate at the GBF and
at the Stanford University (CA, USA) did he work as a scientist at the Caesar Institute
(Bonn, Germany). He then co-founded and worked as senior scientist and applica-
tion specialist at the Biosensor GmbH and now at the SAW Instruments GmbH (both
Bonn).