Surface immobilizable chelator for label-free electrical detection of pyrophosphate

Article abstract of DOI:10.1039/c1cc12073e

A new pyrophosphate (PPi) chelator was designed for surface-sensitive electrical detection of biomolecular reactions. This article describes the synthesis of the PPi-selective receptor, its surface immobilization and application to label-free electrical detection on a silicon-based field-effect transistor (FET) sensor.

Full text of DOI:10.1039/c1cc12073e

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COMMUNICATION
Surface immobilizable chelator for label-free electrical detection
of pyrophosphatew
David J. Liu,*a Grace M. Credo,a Xing Su,a Kai Wu,a Hsiao C. Lim,a Oguz H. Elibol,ab
Rashid Bashirc and Madoo Varma*a
Received 12th April 2011, Accepted 1st June 2011
DOI: 10.1039/c1cc12073e
A new pyrophosphate (PPi) chelator was designed for surface-
sensitive electrical detection of biomolecular reactions. This
article describes the synthesis of the PPi-selective receptor, its
surface immobilization and application to label-free electrical
detection on a silicon-based field-effect transistor (FET) sensor.
detect submicromolar PPi in bulk solution. One class of
chelators is designed such that they can bind to indicator
dyes, where PPi is detected either colorimetrically5 or
fluorescently6 when dye molecules are displaced from chelator
by PPi binding.
Extending chelation-based sensing to surface-sensitive
electrical detection requires a chelator compatible with surface
immobilization and selective to the target analyte. Surface
capture of PPi signaling molecules is expected to enhance the
sensitivity of field-effect devices to PPi in a process that we call
‘‘signal immobilization’’. The negatively-charged PPi molecules
are expected to decrease the number of positively charged
carriers in a p-type field-effect transistor (FET) sensor
functionalized with such a chelator, resulting in a decrease in
threshold voltage. In order to test this ‘‘signal immobilization’’
concept, we designed a new chelator with three functional
components: a binding site, a linker, and a handle. The
binding site selectively captures PPi from solution, while the
linker between the binding site and handle provides steric
flexibility. Finally, the handle ensures that the chelator can be
selectively attached to a chemically compatible surface.
The selected PPi chelator was based on di-(2-picolyl) amine
(DPA), which has demonstrated strong binding affinity to PPi6
and is relatively straightforward to synthesize. The hydroxyl
groups of 5-nitro-1,3-bishydroxymethylbenzene were first
tosylated to accelerate substitution with DPA (Scheme 1).
After DPA substitution, the nitro group was reduced to an
amine by catalytic hydrogenation. The addition of zinc nitrate
produces a functional complex with two Zn2+ coordination
sites per chelator molecule.
Nucleotide incorporation reactions, catalyzed by DNA and
RNA polymerases, are critically important in the biological
processes of living systems. Their common byproduct, pyro-
phosphate (PPi), is a negatively charged small molecule
typically detected using optical techniques, such as chemi-
luminescence.1 Label-free electrical monitoring of biochemical
reactions offers several advantages including increased port-
ability and improved integration.2 The former advantage
is due to the elimination of bulky optical measurement
components and the latter is due to the ability to fabricate
many individually addressable electronic devices at micro- or
nanoscale. Scalable semiconductor manufacturing techniques
can be adapted to produce dense, highly reproducible sensor
arrays to process samples and signals in a highly parallel
fashion. We are developing an electrical signal detection
platform capable of detecting DNA synthesis reactions by
making use of the intrinsic physicochemical properties of PPi.
Here we report the synthesis of a PPi-selective receptor, its
surface immobilization and application to label-free electrical
detection on a field-effect transistor (FET) device.
Various optical PPi detection technologies have been
developed. Among these, luciferase-based PPi detection has
been used for bacterial detection and DNA sequencing
applications.1 Non-enzymatic PPi detection technologies
have also been reported.3,4 These include fluorescence- and
absorption-based detection using PPi chelators, which can
Basic functionality of the synthesized chelator was verified
in solution. Selective binding studies were performed using
a coumarin-based fluorescent dye6 or a colorimetric dye,
pyrocatechol violet (PV).5 In the case of the fluorescent dye,
a Integrated Biosystems Lab, Intel Labs, Intel Corporation,
2200 Mission College Blvd., Santa Clara, CA 95054, USA.
E-mail: djqliu@gmail.com, madoo.varma@intel.com;
Fax: 408-765-2393; Tel: 408-653-9341
b Birck Nanotechnology Center, Purdue University, West Lafayette,
IN 47907, USA
c Departments of Electrical and Computer Engineering and
Bioengineering, Micro and Nanotechnology Laboratory,
University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
w Electronic supplementary information (ESI) available: Synthetic
methods, in-solution assays, surface analysis, and FET data. See
DOI: 10.1039/c1cc12073e
Scheme 1 Synthesis of an immobilizable PPi chelator. (i) TosCl;
(ii) di-(2-picolyl)amine; (iii) H2, Pd/C; (iv) Zn(NO3)2.
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Scheme 2 Surface immobilization of immobilizable PPi chelator.
(i) 4-(Triethoxysilyl)butyraldehyde; (ii) 3, borate (pH 8), NaBHAc3.
Fig. 1 Competitive displacement assay of immobilizable chelator in
solution with PPi ( ), nucleotides dATP ( ), dCTP ( ), dGTP ( ), or
dTTP( ). 1 : 1 mixtures of chelator and pyrocatechol violet (PV) were
treated with the different binders with concentrations ranging from
0–50 mM. Absorption was monitored at 444 nm.
binding to the chelator caused fluorescence quenching. As
more chelator was added, the fluorescence intensity decreased,
reaching a plateau near 10 mM. When the PV was used,
binding to the chelator caused a color change from blue (free
dye, lmax 444 nm) to yellow (complex, lmax 624 nm). The
strong absorption at 624 nm indicates formation of a chelator-dye
complex. This change was also visible to the naked eye. The
dose response curve corresponds to a binding constant of
1.7 Â 106 MÀ1, close to literature values for a similar chelator.6
We further characterized the chelator with a colorimetric
competitive assay. In this assay, PV dye is first treated with an
equimolar amount of chelator to form a 1 : 1 chelator-dye
weak complex. During PPi titration the PV dye is released into
solution because PPi has a higher binding constant. This
process is monitored by UV-Vis spectroscopy. At 444 nm
the absorption increased with PPi concentration, consistent
with dye displacement by PPi (Fig. 1). Similarly the fluorescent
dye was also used to study PPi displacement. In this case, PPi
released the fluorescent dye from the binding site resulting in
increase of its fluorescence intensity at 480 nm when excited at
347 nm. These results also indicated that the addition of an
amine handle to the chelator did not affect its PPi binding
properties. Both fluorescence and absorption data indicate
that the new chelator shows strong selectivity to PPi over
interfering components including nucleotides (such as dATP)
and phosphate (Fig. 1 and the ESIw).
Fig. 2 Surface characterization of chelator on modified silicon and
electrical detection of pyrophosphate with a chelator-modified FET
device. (a) Confirmation of chelator molecule by TOF SIMS
compared to blank and process control samples (green, red lines);
(b) Label-free detection of 25 mM PPi ( ) by a chelator-modified FET
device in pH 8 Tris buffer with Zn2+, compared to overlapping
response in same buffer before PPi exposure (K) and after rinsing
PPi with dilute acid ( ). The inset is an optical microscopy image of the
FET device used for these measurements.
substrate exposed to chelator, but missing the aldehyde
modification (process control, red line).
To demonstrate the effect of ‘‘signal immobilization’’ on PPi
sensitivity, we explored pyrophosphate sensing using a silicon-
on-insulator field effect transistor (SOI-FET) device.7 SOI-FET
devices can sense changes in surface charge and have been used
as a surface sensitive biosensors.2,8–10 Due to the ability to
tune both top gate and back gate voltages, SOI-FET devices
can offer better sensitivity compared to bulk FET devices.11
The chelator-immobilized FET device was exposed to 25 mM
PPi in Tris buffer (pH 8). The drain current versus gate
potential obtained in accumulation mode of the p-type device
shifted towards more negative potentials after exposure to the
PPi-containing solution (Fig. 2b), consistent with a field-effect
caused by the binding of a positively charged molecule to
chelator on the p-type sensor surface. While this response was
not in the expected direction based on charge alone, the
negatively charged PPi was not directly interacting with the
FET sensor surface. We hypothesize that the unexpected
direction of the sensor response may be caused by a surface
dipole effect in which the surface dipole of the chelator is
changed only when the immobilized chelator-zinc complex
binds to PPi at the surface.9,12 A control buffer solution
without PPi containing the same concentration of zinc does
not yield the same effect as exposure to PPi, as shown in
Fig. 2b (‘before’ and ‘after’). In addition, chelator-modified
FET devices were relatively pH insensitive, with similar IV
curves upon exposure to both pH 8 Tris buffer and pH 3
aqueous acetic acid (0.1 M).
The amine-containing PPi chelator can be immobilized onto
many different surfaces provided a proper linker strategy is
devised. To demonstrate that the chelator can be immobilized
to silicon-derived surfaces, we introduced an aldehyde group
to
a clean silicon oxide surface by modification with
4-(triethoxysilyl)butyraldehyde. Compound 3 was covalently
attached to surface by reductive amination using sodium
triacetoxyborohydride (Scheme 2). To ensure the chelator
was indeed immobilized, changes in thin film thickness and
surface properties were characterized by ellipsometry, atomic
force microscope (AFM) and surface-sensitive mass spectro-
metry (TOF-SIMS). Monolayer thicknesses and sample topo-
graphy were consistent with step-by-step surface modification
of silicon surfaces (ESIw). TOF-SIMS measurements of
modified surfaces yielded the expected mass of the immobilized
chelator (Fig. 2a) while the chelator was not detected on a
blank substrate (no reaction, green line) and a negative control
We collected IV characteristics from several devices on the
same chip to characterize PPi selectivity. The devices were
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probed serially after the addition of each solvent. For example,
upon exposure to buffer, four different devices were characterized
in order to avoid drawing conclusions from single, anomalous
devices. Then, the next buffer was introduced to the chamber
and the next set of measurements were collected. Upon
exposure to PPi solution, a similar threshold voltage shift at
1 nA (À0.5 Æ 0.1 V compared to initial buffer) was observed
on four different chelator-modified SOI-FET devices on the
same chip and this response was reversible after a 0.1 M acetic
acid rinse and second incubation in buffer (+0.4 Æ 0.1 V).
While there were slight variations in curve shape and threshold
voltage position from sensor to sensor, the IV curve shapes for
individual sensors for Tris buffer before, during PPi exposure,
and Tris buffer after were similar, as shown in Fig. 2b.
FET devices modified with PEG blocking molecules instead
of PPi-sensitive chelator were not responsive to 25 mM PPi in
Tris buffer solution with Zn2+ (ESIw). In addition, unmodified
FET devices exhibited no response to the same level of PPi in
solution. These observations strongly indicate that PPi capture
by the chelator on the sensor surface causes changes in surface
charge distribution and thus enables selective electrical
detection of PPi. These results also suggested that a chelator-
modified FET device could detect PPi generated from
polymerase reactions in solution. Preliminary data from our
lab indicate this indeed is the case and results will be published
elsewhere.13
also be expanded to other surface-sensitive detection technologies
for PPi or its analogues, such as on-surface optical fluorescent
or colorimetric detection, waveguide-based detection, and
surface plasmon resonance.
We thank J. Zhang, M. Yamakawa, S. Tan, M. Palla and
R. Murray for experimental assistance; J. Daniels for proofreading;
B. Pathangey and A. Proctor (ATD Q&R Lab, Intel) for
providing TOF SIMS data; B. Reddy, Jr. and the staff at the
Micro and Nanotechnology Laboratory at UIUC for FET
processing; and Stanford Nanofabrication Facility (SNF) for
providing instrument access for ellipsometry.
Notes and references
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T.-W. Tsai, B. R. Dorvel, J. Daniels, R. Bashir, M. Varma, 2011,
submitted.
In summary, we have synthesized and characterized a new
type of pyrophosphate chelator and demonstrated electronic
detection of PPi by immobilizing it to a field-effect sensor.
Because PPi is a common product of many important
biological reactions,14 the concept of ‘‘signal immobilization’’
of PPi presented in this work can be applied to develop
electronic biosensors for broad biomedical applications from
DNA sequencing to microbe detection. This concept could
14 J. K. Heinonen, Biological role of inorganic pyrophosphate, Kluwer
Academic Publishers, London, U.K, 2001, ch. 1, pp. 1–28.
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