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Label-Free Electrochemical Recognition of DNA Hybridization by Means of Modulation of the Feedback Current in SECM.

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Scanning Probe Techniques
Label-Free Electrochemical Recognition of DNA
Hybridization by Means of Modulation of the
Feedback Current in SECM**
Florin Turcu, Albert Schulte, Gerhard Hartwich, and
Wolfgang Schuhmann*
Carrying systematically ordered libraries of gene-specific
nucleic acid recognition entities on solid supports, DNA
microarrays (“DNA chips”) are microanalytical tools for
studying the activity of a variety of genes at a time. Such DNA
microarrays are thus valuable for rapid gene-expression
profiling, sequence mapping, and genotyping of mutations
and polymorphisms or pharmacogenomics.[1] Hybridization of
immobilized, single-stranded DNA capture probes with
complementary DNA fragments in solution (targets) has to
be efficiently transformed into a detectable analytical signal
for reliable application of DNA microarrays. Electrochemical
(EC) detection schemes[2] were explored and considered costeffective alternatives to well-established fluorescence-based
optical read-outs of hybridization as they offer a high
sensitivity in combination with simplicity of instrumentation
and compatibility with microfabrication technology. In general, EC detection schemes take advantage of inherent
electrochemical properties of single stranded (ss) and
double stranded (ds) DNA and/or electrochemically active
hybridization indicators, intercalating compounds, and redox
Herein, we report an electrostatic approach for visualizing
the status of surface-bound DNA probes. Detection of
complementary DNA hybridization is simply achieved
through coulomb interactions between a negatively charged,
free-diffusing redox mediator and the phosphate groups in the
backbone of the immobilized DNA strands. Electrostatic
repulsion between the two ionic species is capable of
modulating the transfer of mediator molecules towards the
electrode surface at DNA-modified regions. A corresponding
local modulation of current was used to establish a simple and
sensitive assay for truly label-free electrochemical imaging of
single-stranded capture probes and their aggregates with
complementary targets.
[*] Dipl.-Chem. F. Turcu, Dr. A. Schulte, Prof. Dr. W. Schuhmann
Analytische Chemie—Elektroanalytik & Sensorik
Ruhr-Universit9t Bochum
44780 Bochum (Germany)
Fax: (+ 49) 234-321-4683
Dr. G. Hartwich
FRIZ Biochem
Staffelseestrasse 6, 81477 Munich (Germany)
[**] The authors would like to thank Dr. Thomas KratzmDller, FRIZ
Biochem, Munich (Germany) for spotting the samples and
comparative fluorescence measurements as well as Dr. Herbert
Wieder, FRIZ Biochem, Munich (Germany) for helpful discussion.
We are grateful to the DFG for financially supporting this work (DFG
Schu929/6-1). SECM = scanning electrochemical microscopy.
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
A DNA microarray is a sample with considerable local
variations in surface redox activity, in particular when anionic
electroactive species are used. For imaging these regions and
for verifying the above-mentioned concept of electrostatic
detection of hybridization, scanning electrochemical microscopy (SECM)[4] was the method of choice. SECM has an
exceptional capability to visualize microscopically small local
variations in (electro)chemical reactivity with high spatial
resolution. Furthermore, SECM has demonstrated a high
level of performance for studying immobilized biomolecules
and biological processes at solid–liquid interfaces.[5] Highprecision positioning devices are used in SECM to position a
disk-shaped voltammetric ultramicroelectrode (the SECM
tip) within the regime of electrochemical feedback, typically
at working distances of only a few electrode radii. In the
simplest mode of operation, the SECM tip is scanned in
constant height above the sample, while simultaneously
monitoring the tip current (I) as a function of x/y tip position.
The dependence of the microelectrode response on the tip-tosample distance (d) and on the nature of the substrate allow
revealing spatially resolved information about chemical
properties and the morphology of the surface.
Figure 1 shows the suggested repelling mode of SECM as
used to recognize the presence, and to characterize the status
Figure 1. Schematic representation of repelling-mode SECM. A) A diffusion-limited steady-state reduction current is observed in
[Fe(CN)6]3 -containing solutions with the tip kept at 0 mV (vs. Ag/
AgCl/3 m KCl). B) Recycling of tip-generated [Fe(CN)6]4 to [Fe(CN)6]3
occurs with the SECM tip close to a propane thiol-modified Au surface
causing an increase in tip current because of positive feedback redox
cycling. C) Above a DNA spot, tip-generated [Fe(CN)6]4 ions experience charge interactions with the oligonucleotide backbone causing a
hindered diffusion of the mediator to the Au surface and hence a modulation of the mass-transfer rate which provokes a drop in tip current.
D) Hybridization increases the density of anionic phosphate groups
leading to a further drop in tip current owing to enhanced repulsion of
the redox mediator molecules.
(ss/ds) of immobilized DNA. The SECM tip is kept at
sufficient constant cathodic potentials to reduce [Fe(CN)6]3
under diffusion control. Far above the sample surface, the
amperometric tip current is controlled by the diffusion of
[Fe(CN)6]3 molecules towards the tip, which leads to a
steady-state current (I0, Figure 1 A). However, after approach
of the SECM tip to nearfield distance to a conducting surface
(propane thiol-modified Au), consumed mediator molecules
can be reconverted into their initial oxidation state (electro-
DOI: 10.1002/anie.200454228
Angew. Chem. Int. Ed. 2004, 43, 3482 –3485
chemical recycling, positive feedback)[4] in turn leading to a
distance-dependent increase in the tip current (IAu > I0,
Figure 1 B). Above a DNA spot, the anionic phosphate
groups repel the tip-generated [Fe(CN)6]4 molecules. This
process hinders their diffusion towards the Au electrode,
reduces the rate of recycling and leads to a local drop in the
observed cathodic tip current (Iss < IAu, Figure 1 C). The
hybridization of the immobilized capture-probe strands with
complementary single strands and the resulting formation of
double helices multiplies the number of negative charges on
the surface, which leads to a further drop in tip current (Ids <
Iss, Figure 1 D).
SECM measurements were performed on 150-mm diameter spots of bifunctionalized 20-base synthetic oligodeoxynucleotides using a 10-mm diameter Pt microdisk electrode as
the SECM tip. The 3’ thiol-modified DNA strands with a
fluorescent label attached to the 5’ end were immobilized on
gold-covered glass slides through standard thiol-monolayer
assisted molecular self-assembly. To avoid critical nonspecific
adsorption and to increase hybridization efficiency, surfaces
were treated with propane thiol after spotting with oligonucleotides. In the electrochemical cell of the SECM, DNAmodified slides were covered with an electrolyte containing
5 mm K3[Fe(CN)6] in 3 m NaCl/0.1m phosphate buffer (1:1
KH2PO4/K2HPO4 ; pH 5.7). Far away from the chip surface
and operated amperometrically at a potential of 0 mV versus
Ag/AgCl/3 m KCl, the Pt microelectrode typically displayed a
steady-state reduction current (I0) of about 6 nA owing to
the diffusion-controlled reduction of Fe3+ centers to Fe2+
centers. Before imaging capture-probe spots, tip approach
curves (I as a function of d) were recorded in the neighborhood of the spots and used to determine the vertical
expansion of positive feedback and hence the chip-to-tip
distance. Based on this information and theoretically derived
approach curves,[6] the microelectrode tip could be positioned
reproducibly and scanned across the sample at a user-defined
spacing. Line scans (I as a function of traveled distance in the
x direction) were acquired for the spatially resolved analysis
of the DNA-modified surface.
In Figure 2, curve 1 displays a representative amperometric recording taken during a complete line scan over an
individual oligonucleotide spot. The current values to the left
and right of the spot are indicative for the unbiased positive
feedback from the propane thiol modified gold surface. In
agreement with the model, drastically lowered current values
in the central part (above the DNA spot) signify reduced rates
of redox recycling owing to electrostatically hindered diffusion. Note that there was almost no drop in the tip current
when the negatively charged mediator was substituted by a
positively charged mediator. With [Ru(NH3)6]3+, only a
negligible decrease in current occurs (Figure 2, curve 2)
which is probably caused by the purely steric influence of
the surface-bound capture probes on the mass-transfer rate.
This effect is proof that when measuring in the presence of a
negatively charged mediator an electrostatic repellent force
really is responsible for a DNA-induced modulation of the tip
High-quality, 3D SECM images (I vs. x/y) of DNA spots
were constructed from multiple line scans (Figure 3). The
Angew. Chem. Int. Ed. 2004, 43, 3482 –3485
Figure 2. Line scans acquired by scanning the tip of a 10-mm-diameter
Pt microelectrode at a fixed height of 10 mm across a spot of the
single-stranded capture oligonucleotide. 1) [Fe(CN)6]3 ions (5 mm in
3 m NaCl/0.1 m phosphate buffer (1:1 KH2PO4/K2HPO4) at pH 5.7)
were used as the mediator with the SECM tip being polarized to 0 mV
(vs. Ag/AgCl/3 m KCl). 2) [Ru(NH3)6]3+ ions (5 mm in 3 m NaCl/0.1 m
phosphate buffer; pH 5.7) were used as mediator with the SECM tip
being polarized to 400 mV (vs. Ag/AgCl/3 m KCl).
Figure 3. Three-dimensional SECM image of an individual spot of a
single stranded 20-base oligonucleotide as obtained by means of
repelling-mode SECM. Solution: 5 mm [Fe(CN)6]3 in 3 m NaCl/0.1 m
phosphate buffer (pH 5.7); tip potential: 0 mV (vs. Ag/AgCl/3 m KCl).
images demonstrate the potential of the repelling mode of
SECM for imaging immobilized nucleic acids. Such images
can be used for controlling the quality of the spotting process
and the homogeneity of the lateral surface concentration of
the DNA capture probes within the spot. To test the
suitability of the approach as a hybridization assay, spots of
ssDNA were subjected to hybridization experiments before
SECM imaging. One of two neighboring spots was exposed to
a diluted solution of the complementary target DNA while
the other (control) was covered with target-free “hybridization solution”. Upon completion of hybridization,[7] slides
were rinsed and the two spots were examined by means of
repelling-mode SECM.
Figure 4 illustrates the result obtained, and that hybridization could be clearly detected as a visible difference
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
capture probes, length of targets, temperature, ionic strength,
and pH value of the scanning solution) on the performance of
the method.
Experimental Section
Figure 4. Detection of hybridization through electrostatic repulsion
and visualization of DNA duplex formation by means of repellingmode SECM. A selected spot of a 20-base oligonucleotide was exposed
to the hybridization buffer (1 m NaCl/0.1 m phosphate buffer; pH 6.3)
containing 2 mm of the complementary oligonucleotide target. A neighboring spot was subjected to target-free hybridization buffer (control).
After completion of hybridization (about 2 h) and thorough rinsing
with 3 m NaCl/0.1 m phosphate buffer (pH 5.7), SECM line scans were
measured by operating the tip of a 10-mm-diameter Pt microelectrode
(0 mV vs. Ag/AgCl/3 m KCl) in solutions of 5 mm [Fe(CN)6]3 in 3 m
NaCl/0.1 m phosphate buffer; pH 5.7).
between the response of the SECM tip above the control and
hybridized spot. Consistent with our hypothesis, the cathodic
tip current was found to be much lower at the hybridized spot
since here duplex formation took place concomitantly
increasing the number of repellent charges.
The detection scheme described herein was performed by
taking local electrochemical measurements in a sophisticated
SECM set-up that clearly is not suitable for common use in
medical diagnostics. However, in principle, it should be
possible to design a simplified electrochemical device that
could be produced using microfabrication technology. Such a
device could, for example, consist of an array of immobilized
DNA capture probes in a base plate and an array of
individually addressable Pt microelectrodes in a cover plate.
Using specially designed spacer and alignment elements, the
microelectrodes and DNA spots could be arranged on top of
each other and kept at the proper working distance. The
difference in the response of an individual Pt microelectrode
before and after exposure to a sample would evaluate the
hybridization status of the DNA capture probes in the
corresponding region.
In summary, we confirmed that simple coulomb interactions between immobilized DNA and ferricyanide effectively modulates the diffusion transport properties of the
dissolved mediator to the gold surface. This property has a
strong impact on local amperometric feedback currents and
allows the imaging of DNA-modified surface spots by SECM.
The increase in the density of negative charge as a result of
duplex formation made electrostatic repulsion a sensitive
assay for truly label-free electrochemical recognition and
visualization of hybridization. For optimization and quantification, we are currently studying the influence of a number of
parameters (i.e. surface density and lengths of the DNA
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Spotted DNA microarrays, oligonucleotides probes, and targets were
supplied by FRIZ Biochem, Munich. Probes were 3’ thiol modified
20-mer oligonucleotides ([Fluo-HN-C12]-TGC GGATAACAC AGTCAC CT-[C3-S-S-C3-OH]) while as target an unmodified oligonucleotide with the complementary sequence (ACG CCTATT GTG TCAGTG GA) was used. Spotting of the probe was on Au-sputtered
glass slides using a microarrayer (Cartesian Technologies) equipped
with a printhead (ChipMaker) and microspotting pins (120 mm
diameter; Telechem International). The capture probes were arrayed
in spots of 150-mm diameter in two rows with a fine scratch dividing a
control area from a hybridization area. An O-ring defined the area
subjected to SECM measurements.
Solutions required for the experiments were prepared with tridistilled water. KH2PO4, K2HPO4·3 H2O, NaCl, K3[Fe(CN)6], and
[Ru(NH3)6]Cl3 were purchased from Sigma. SDS (Sodium dodecylsulphate) was from Merck.
Conventional, glass-insulated Pt disk microelectrodes (1 10 mm,
RG values 10–20) were used as SECM tips. They were polished with
alumina paste (particle sizes: 3, 1, and 0.3 mm) on a soft polishing
cloth and tested by cyclic voltammetry in 5 mm ferricyanide solutions.
SECM experiments were performed with a home-built SECM in a
one-compartment cell and with a two-electrode configuration (the
SECM tip and a miniaturized Ag/AgCl 3 m KCl reference electrode).
The set-up was located in a Faraday cage and a low-noise amplifier
(VA10, npi electronic) was applied for the electrochemical measurements. The electrochemical cell with the sample fixed to the bottom
was mounted on a two-axis translation stage driven by computercontrolled stepper motors (Owis), which allowed sample movements
in the x, y direction with a nominal resolution of 0.625 mm per half
step. Tip approach (z movement) was achieved with a third stepper
motor that was mounted perpendicular to the ones moving the cell. A
PC in combination with Windows software programmed in Microsoft
Visual Basic 3.0 (Microsoft) was used for the control of all system
parameters and for data acquisition (16 bit AD/DA card; Plug-In).
Tip approach curve measurements (z scans) helped to position
the SECM tip within the nearfield of electrochemical feedback. The
working distance was chosen to a value corresponding to an increase
in the amperometric tip current of 50 % of the value measured in bulk
solution and typically was about 10 mm. SECM imaging was achieved
by moving the DNA chip at fixed tip-to-sample separation in x, y
direction while simultaneously monitoring the tip current as a
function of position.
Received: March 10, 2004 [Z54228]
Published Online: June 8, 2004
Keywords: DNA recognition · electrostatic interactions ·
scanning probe techniques · SECM · surface analysis
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2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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labe, current, free, mean, feedback, dna, hybridization, secm, recognition, modulation, electrochemically
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