A parallel microfluidic channel fixture fabricated using laser ablated plastic
laminates for electrochemical and chemiluminescent biodetection of DNA
Thayne L. Edwards, Jason C. Harper, Ronen Polsky, DeAnna M. Lopez, David R.
Wheeler, Amy C. Allen, and Susan M. Brozik*
Biosensors & Nanomaterials, Sandia National Laboratories, PO Box 5800, MS-0892, Albuquerque, NM
87185 USA
Telephone: (505) 844-5105; Fax: (505) 845-8161; Email:smbrozi@sandia.gov
Supplemental Information
Electrografting of Aryl Diazonium Salts in Ti/Au Fixture
Figures S1-S6
Electrochemical DNA Detection Assay
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Electrografting of Aryl Diazonium Salts in Ti/Au Fixture
Selective functionalization of individual electrodes in the microfluidic fixture was
realized via bias-assisted grafting of aryl diazonium salts. The electrochemical reduction
of an aryl diazonium salt creates an aryl centered radical with elimination of dinitrogen.1
These aryl radicals can then react with conducting or semiconducting surfaces forming a
covalent bond. While this is a simplified description of the grafting, and the nature of this
bond with Au substrates is still under investigation, the resulting thin film is highly
stable, withstanding several minutes of ultrasonication and long-term storage in air while
remaining conductive, which is critical for subsequent electrochemical measurements.2
Bias-assisted deposition of carboxylphenyl and nitrophenyl diazonium onto the
thermally evaporated Ti/Au working electrodes (WEs) in the fixture (using the common
on-chip Au pseudo-reference and Pt counter electrodes) was studied using voltammetry.
Cyclic voltammograms of the respective diazonium salts are presented in Figure S1. A
sharp irreversible peak and a broad cathodic wave was observed on the first scan with
carboxylphenyl diazonium at Ep,c = -180 mV and -540 mV, respectively, vs. the Au
pseudo-reference (see Figure S1A, black trace), and are attributed to the reduction of the
diazonium salt. Multiple reduction peaks for diazonium reduction have been observed on
Au electrodes and have been attributed to reduction through different crystallographic
phases on the Au surface.3 Both peaks were significantly suppressed and negatively
shifted by ~100 mV on the second CV scan (Figure S1A, grey trace). The higher
overpotential required for reaction and lower relative currents on the second scan are
characteristic of electrochemistry at films derived from diazonium electrodeposition, and
are a manifestation of the higher resistance to electron transfer through the phenyl thin
film. Nitrophenyl diazonium electrodeposition yielded two sharp irreversible peaks at Ep,c
= -160 mV and -410 mV (Figure S1B, black trace). No Faradic currents were observed
on the subsequent CV scan (Figure S1 B, grey trace) and the overall current response was
significantly suppressed, again characteristic of thin film formation via bias-assisted
grafting.
The electrodeposition protocol used to form the grafted thin film has been shown
to have a profound affect on the order, thickness, and electron transfer kinetics of the
resulting film.4 Previous results have shown that fix potential depositions lead to thinner
2
and more ordered films, allowing for improved current responses, than potential sweep
methods which produce thicker films that can hinder electron transfer.4 Therefore,
chronoamperometric electrodeposition was used to modify WEs with thin films to
immobilize ssDNA probes (see Experimental Methods section for parameters). Figure
S1, C and D show the current responses with time for carboxylphenyl diazonium
deposition at 6 Au WEs, and nitrophenyl diazonium deposition at 3 Au WEs,
respectively, in a 9 WE microfluidic fixture. An initial period of high cathodic current is
observed corresponding to diazonium reduction and double layer charging. The current
response then decreases as the thin film forms and resistance to electron transfer
increases. A low degree of variability in current response during electrodeposition
between all electrodes on the array strongly suggests consistent deposition of the
diazonium salt across all electrodes.
REFERENCES
1. (a) M. Delamar, R. Hitmi, J. Pinson, and J.-M. Savéant, J. Am. Chem. Soc. 1992, 114,
5883–5884. (b) M.-C. Bernard, A. Chaussé, E. Cabet-Deliry, M. M. Chehimi, J.
Pinson, F. Podvorica, and C. Vautrin-Ul, Chem. Mater. 2003, 15, 3450-3462.
2. (a) G. Z. Liu, T. Böcking, and J. J. Gooding, J. Electroanal. Chem., 2007, 600, 335344. (b) A. Laforgue, T. Addou, and D. Bélanger, Langmuir 2005, 21, 6855-6865. (c)
J. C. Harper, R. Polsky, D. R. Wheeler, and S. M. Brozik, Langmuir 2008, 24, 22062211.
3. A. Benedetto, M. Balog, P. Viel, F. Le Derf, M. Sallé, and S. Palacin, Electrochim.
Acta, 2008, 53, 7117-7122.
4. (a) J. C. Harper, R. Polsky, S. M. Dirk, D. R. Wheeler, and S. M. Brozik,
Electroanalysis 2007, 19, 1268-1274. (b) H. Uetsuka, D. Shin, N. Tokuda, K. Saeki,
and C. E. Nebel, Langmuir 2007, 23, 3466-3472. (c) J. C. Harper, R. Polsky, D. R.
Wheeler, S. M. Dirk, and S. M. Brozik, Langmuir, 2007, 23, 8285-8287. (d) J.
Haccoun, C. Vautrin-Ul, A. Chaussé, and A. Adenier, Prog. Org. Coat. 2008, 63, 1824. (e) J. C. Harper, R. Polsky, D. R. Wheeler, D. M. Lopez, D. C. Arango, and S. M.
Brozik, Langmuir, 2009, 25, 3282-3288.
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Figure S1
Figure S1. Cyclic voltammograms of A) 1 mM carboxylphenyl diazonium and B) 1 mM
nitrophenyl diazonium at unmodified 500 M diameter Au WEs with on-chip Au
pseudo-reference and Pt counter electrodes in 0.1 M Bu4NBF4, in ACN, v = 100 mV s-1.
Initial (black) and second (grey) CV scans are shown. Chronoamperometric
electrodeposition of C) carboxylphenyl diazonium at 6 of the 9 Au WEs and D)
nitrophenyl diazonium at 3 of the 9 Au WEs on the microfluidic electrode array. 1mM
diazonium salt in 0.1 M Bu4NBF4, in ACN, applied potential = -1.0 V vs. Au pseudoreference.
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Figure S2
Figure S2. Cyclic voltammograms of 1 mM Fe(CN)63-/4- in 0.1 M KCl, v = 100 mV∙s-1, at
each of nine individually addressable 500 m diameter Au electrodes vs. on-chip
common Au pseudo-reference, and shared Pt counter. The second CV scan is shown.
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Figure S3
Figure S3. Cyclic voltammograms of 1 mM Fe(CN)63-/4- in 0.1 M KCl, v = 100 mV∙s-1, at
each of nine individually addressable 500 m diameter ITO electrodes vs. on-chip
common Au pseudo-reference, and shared Pt counter. The second CV scan is shown.
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Figure S4
Figure S4. Cyclic voltammogram of 5 mM ruthenium bipyridine, 50 mM tripropylamine
in 1x PBS, pH 7.4, at an ITO working electrode in a plastic laminate fixture. On-chip Au
pseudo-reference and Pt counter electrodes; v = 100 mV/s.
Figure S5
Figure S5. Plot of electrochemiluminecent reaction light intensity imaged through the
underside of an ITO electrode vs. applied potential during a cyclic voltammetry sweep
from 0 to 2 to 0 volts vs. the on-chip Au pseudo-reference electrode. 5 mM ruthenium
bipyridine, 50 mM tripropylamine in 1x PBS, pH 7.4. On-chip Pt counter; v = 100 mV/s.
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Figure S6
Figure S6. Electrochemical DNA Detection Assay
Electrochemical DNA Detection Assay
Array channels were treated with 10 M of ssDNA or dsDNA sequences 2 (breast
cancer target), 5 (colorectal cancer target), and/or 7 (random control) in 2x SSC for 2
hours at room temperature. Following the 2 hour treatment with analyte solution, the
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channel was thoroughly rinsed with 2× SSC, resulting in a surface with bound target
DNA (Figure S6, step 1). This surface was then treated with 10 M of biotinlyated DNA
detection sequence 3 and 6 together (detection probes) in 2× SSC solution for 2 hours at
room temperature. These detection sequences will bind to the surface only if target DNA
sequences were present in step 1 and bound to the electrode surface. After treatment, the
array was rinsed twice with 2× SSC solution (Figure S6, step 2). The array channels were
then incubated with a 1:1000 dilution of ExtrAvidin-HRP (electrochemical label, HRP =
horseradish peroxidase) in peroxidase stabilizer solution for 45 minutes. Afterwards, the
channels were rinsed with 1× PBS, pH 7.4 (Figure S6, step 3). For detection, a given
channel was exposed to TMB conductivity solution, which contains HRP substrate
(H2O2) and TMB mediator (Figure S6, step 4).
The chronoamperometric current at each working electrode was measured at -300
mV vs. the common Au pseudo-reference. The current measured is a function of bound
HRP catalysis, with TMB serving as an electron mediator between the Au electrode and
the heme cofactor of the redox active enzyme.
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