Download Supporting Information

Supporting Information
This file gives additional information including description of the microfluidic chamber,
discussion on the fluidic parameters, XPS analyses, complementary impedance measurements
and characterization of sensor's kinetics.
A Microfluidic Electrochemical Biosensor based
on MWCNT/ferrocene for Genomic DNA
Detection of Mycobacterium Tuberculosis in
clinical isolates
Bacem Zribi,a,b,c Emmanuel Roy,a Antoine Pallandre,a,d Syrine Chebil,a Mohamed Koubaa,c
Nawel Mejri,b Harrison Magdinier Gomes,e Christophe Sola,e Hafsa Korri-Youssoufi‡b and
Anne-Marie Haghiri-Gosnet‡*a
a.
Laboratoire de Photonique et de Nanostructures (LPN), CNRS, Université Paris
Saclay, route de Nozay, 91460 Marcoussis, France.
b.
UMR-CNRS 8182, ICMMO, Université Paris-Sud, Bat 420, 91405 Orsay, France.
c.
Laboratoire de Physique des Matériaux, LPM FSS, Faculté des Sciences de Sfax,
Tunisie.
d.
Université Paris Sud, Faculté de Pharmacie, 5 rue Jean-Baptiste Clément, 92290,
Châtney-Malabry, France.
e.
Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud, Bat 400,
91405 Orsay, France.
SI.1 Size of the microfluidic chamber
The various sizes of both the different electrodes and microfluidic chamber are shown in the
following figure.
Figure SI.1. (a) Schematic
view of the microfluidic electrochemical cell and (b) SEM image
shows a cross-section of the 1.6 μm-thick CNT layer.
SI.2 Fluidic parameters
Herein, we proposed a flow pulsed microfluidic approach to improve mass transport that
contributes to enhanced target-probe interaction events. For most of applications, if the fluidic
system works under “extreme fast” flow at a high Péclet number PeH, the number of captured
molecules per unit of time is largely enhanced.
Figure SI.2. The pulsed fluidic protocol based on a first dynamic step at high PeH during t1
producing a thin depletion zone (S=0.23 µm) for extremely fast trapping of DNA targets
followed by a conventional static diffusion regime during t2.
To determine in microfluidic devices whether analytes are depleted in the vicinity of the
sensor surface and which limiting factors govern kinetics, several parameters have to be
considered. The geometry of the fluidic channel (the height H and the width W), the size of
the sensor (the width WS and the length LS) and the volumetric flow rate Q have thus to be
precisely defined to fix if the system will work in a reaction-limited or diffusion-limited
regime. In 2008, Squires, Messinger and Manalis have proposed a theoretical approach to
estimate the interplay between diffusion, convection and reaction.
1
Their complete analysis
gives some guidelines to estimate some fundamental quantities, such as fluxes, collection
rates and equilibration times. Based on dimensionless parameters that are straightforward to
compute, their simple approach is very useful in designing optimized systems. At low values
of PeH (PeH ≪ 1 at extremely slow flow), the system is in a diffusive regime, with a
depletion zone that extends far upstream in the whole thickness of the channel. Even if all
molecules are collected, the time of capture is so high (hours or days) that such diffusive
regime appears very limited for real applications. At high PeH (PeH ≫1 at extremely fast
flow), the depletion zone (S) becomes thinner than the channel, which enhances the number
of molecules trapped in the vicinity of the sensor surface (Figure SI.2).
First, the two Péclet numbers have to be estimated, namely 𝑃𝑒𝐻 = 𝑄 ⁄𝐷 × 𝑊 with subscript
𝐻 to specify the channel height as the relevant parameter and 𝑃𝑒𝑠 = 6 (𝐿/𝐻)2 𝑃𝑒𝐻 , where 𝐷
is the diffusion rate, 𝑄 is the volumetric flow rate, 𝑊 is the channel width and 𝐿𝑆 is the sensor
length. Our sensor with 𝐿𝑆 = 900 𝜇𝑚 and 𝑊𝑆 = 1323 𝜇𝑚 is integrated in the microfluidic
channel with 𝐻 = 60 𝜇𝑚 and 𝑊 = 1523 𝜇𝑚 through which a target solution flows at a flow
rate 𝑄 = 150 𝜇𝑙 ⁄𝑚𝑖𝑛. Our microfluidic system exhibits large values of both Péclet numbers:
-
-
For Hepatitis C DNA target containing 15 base pairs (at C0 = 7.5 fM), with
D0 = 4 10-2 µm2/s, the two Péclets numbers are: 𝑃𝑒𝐻 = 4.1 × 107 and 𝑃𝑒𝑠 = 5.5 ×
1010 which means that the depletion zone is largely thinner than the channel and the
sensor:
1
𝛿𝑆 ~ 𝐿𝑆 ⁄(𝑃𝑒𝑠 ) ⁄3 = 230 𝑛𝑚 ≪ 𝐿 < 𝐻
For M. Tuberculosis DNA target containing 411 base pairs, with D0 = 4 10-4 µm2/s, the
two Péclets numbers are 𝑃𝑒𝐻 = 4.1 × 105 and 𝑃𝑒𝑠 = 5.5 × 108 which gives:
𝛿𝑆 = 1.09 µ𝑚 ≪ 𝐿 < 𝐻
As expected, the extremely fast flow JD produces a very thin depletion zone at the surface of
the sensor favoring a maximum value of collection rate of target molecules.
The flux JD of target molecules reaching the device can be approximated following this
equation2,3 𝐽𝐷 ~ 𝐷𝐶0 𝑊𝑠 𝐹, where the dimensionless mass transport flux is calculated according
this equation 𝐹~𝑃𝑒𝑠 1/3 . For Hepatitis C DNA target with C0 = 7.5 fM, 𝐽𝐷 = 0.91 molecule
per second. Finally, one should then estimate the Damkohler number that indicate how
equilibrium will be reached from 𝐷𝑎 = (𝑐0 − 〈𝑐〉𝑆 )⁄〈𝑐𝑆 〉 = 𝑘𝑜𝑛 𝑏𝑚 𝐿⁄𝐷𝐹 , where C0 and <
Cs> are the bulk and average concentration at the sensor surface, 𝑘𝑜𝑛 ~106 𝑀−1 𝑠 −1 (standard
value for a DNA target) and 𝑏𝑚 = 2 × 1011 𝑐𝑚−2 (the previously reported value for specific
surface of CNTs).
3
When Da >> 1, the kinetics of hybridization become mass-transport
limited, while at Da << 1, the transport becomes limited by the reaction. For Hepatitis C DNA
target, 𝐷𝑎 = 19.6. Furthermore, the number of DNA strands collected can be estimated. For
Hepatitis C DNA target, at very low concentration C0 = 7.5 fM the use of dynamic mode
enables to capture 55 targets in 1 min (JD × 60) at 150 µl/min, whereas the use of static mode
applied for 9 min allows to capture only 14 targets in the volume 𝑉 ∗ = 3 𝑛𝐿 which
corresponds to the diffusion height at the sensor surface 𝐻 ∗ = √𝑡 ∗ × 𝐷 = 2.2 µm. The whole
pulsed protocol during 40minutes (4x10min) collect 272 targets at this very low concentration
of 7.5 fM.
Table SI.2 Microfluidic system (dynamic mode).
Q=150µl/min
Hepatitis C
C0= 7 fM
Tuberculosis
D (10-2 µm2s-1)
4
4 10-2
PeH= 𝑄⁄𝐷 × 𝑊𝑐
PeS= 6 (𝐿/𝐻)2 𝑃𝑒𝐻
1
𝛿𝑠 ~ 𝐿⁄(𝑃𝑒𝑠 ) ⁄3
F ≈ 0.81 𝑃𝑒𝑠 1⁄3
JD= 𝐷𝑐0 𝑊𝑆 𝐹
Da
4.1 107
5.5 1010
0.23 µm
3812
0.91 molecules s-1
19.6
4.1 105
5.5 108
1.1 µm
819
References
1
T.M. Squires, R.J. Messinger, and S.R. Manalis, Nature Biotechnology 26, 417 (2008).
2
J.L. Arlett, E.B. Myers, and M.L. Roukes, Nature Nanotechnology 6, 203 (2011).
3
M.R. Leyden, R.J. Messinger, C. Schuman, T. Sharf, V.T. Remcho, T.M. Squires, and E.D.
Minot, Lab on a Chip 12, 954 (2012).
SI.3 X-Ray Photoelectron Spectroscopy analysis
All the different chemical steps have been characterized by XPS (Figure SI.3). After the
grafting of the amine, the nitrogen peak (N1s) recorded with height resolution shows clearly
the presence of amine functions at 399.9eV. After ferrocene grafting, two peaks
corresponding to Fe2 + and Fe 3 + will take place respectively at 720.8 eV and 708 eV. Finally,
the grafting of ssDNA is confirmed by the appearance of a peak at 133.6 eV, which can be
attributed to phosphate ions. After modification of the surface with EDA molecules, the
deconvolution of the corresponding nitrogen peak has been performed using the Shirley-type
baseline and Lorentzian-Doniac-Sunsic curves, with Gaussian profile. The fit shows the
appearance of two nitrogen peaks at 399.90 eV and 402.11 eV which could be respectively
attributed to C-N link (84 %: atomic proportion) and free amine NH2 (16 %: atomic
proportion).
Figure SI.3. XPS spectra of MWCNTs recorded after physical adsorption of the CNTs on the gold electrode and
after the electrochemical patterning of the probe “EDA-Fc-ssDNA”.
SI.4 EIS impedance measurements
The electrical properties of each patterned layer (Au, Au-MWCNT, Au-MWCNT-EDAFc(NHP), Au-MWCNT-EDA-Fc(NHP)-ssDNA and Au-MWCNT-EDA-Fc(NHP)-dsDNA)
after each functionalization step inside the fluidic chip were examined with electrochemical
impedance spectroscopy in the presence of an external redox marker [Fe(CN)6]4-/[Fe(CN)6]3(10 mM in KCl) (Figure SI.4).
Figure SI.4. Nyquist plots obtained in the frequency range from 0.5 Hz to 10 kHz in 10 mM [Fe(CN)6]3-/4- by
applying AC potential (10 mV). The symbols are the experimental data and the solid are the fitted curves using
the equivalent circuit showed in inset.
Nyquist plots show a semi-circle fitted by Randles equivalent circuit model where Rs is the
electrolyte resistance (900 Ω), Rct is the electron transfer resistance, and CPE is a constant
phase element.
The role of ferrocene in the tunneling of charge carriers between NTCs contacts and DNA
strand(s) is also of great importance. After the covalent immobilization of single-strand
oligonucleotides on ferrocene our SIE measurements evidence a decrease of Rct and thus a
more metallic character of the NTCs/ferrocene/ssDNA transducer (see Fig. SI.4) is obtained.
This phenomenon is due to disturbance of the electron cloud of the ferrocene molecule
induced by the effect of negative charges acquired by the ions of the DNA phosphate groups.
Once hybridization occurs, Rct increases largely, reflecting a less metallic behavior due to the
emergence of another competitive phenomenon with charge consumption. 4 The hybridization
phenomenon thus results in a reduction of the charge transfer rate causing an increase in Rct
as observed in our experimental measurements.
The experimental data from the Nyquist plots recorded at each chemical step have been
fitted by Randles equivalent circuit model to provide details in the kinetics of charge transfer
through the sensing layer occurring at the integrated electrodes in the fluidic chip. The Rct,
CPE1, CPE2 values obtained are listed in the table below. The obtained error values χ2 are in
the order of 10-2 which confirms that the modified equivalent circuit model for fitting is well
adapted.
Rct (103 Ω)
CPE1 (10-6 Mho)
CPE2 (10-6 Mho)
χ2
5.87 ± 0.11
0.34 ± 0.04
101 ± 4.135
0.03
4.81 ± 0.17
N=0.678 ± 0.01
1.7 ± 0.19
N=0.537 ± 0.02
166 ± 4.86
0.01
Au-MWCNTs-Fc
1.44 ± 0.09
N=0.574 ± 0.01
0.12 ± 0.03
N=0.398 ± 0.02
119 ± 2.17
0.03
Au-MWCNTs-Fc-ssDNA
0.541 ± 0.12
N=0.793 ± 0.03
0.06 ± 0.04
N=0.33 ± 0.01
114 ± 1.6
0.03
Au-MWCNTs-Fc-dsDNA
2.42 ± 0.16
N=0.875 ± 0.06
0.571 ± 0.12
N=0.272 ± 0.01
138 ± 7.16
0.02
N=0.698 ± 0.02
N=0.317 ± 0.01
Au
Au-MWCNTs
Table SI.4. The fitted data from the Randles equivalent circuit model Rct, CPE1, CPE2 and
the overall fitting error χ2.
Reference
4
D.M. Adams, L. Brus, C.E.D. Chidsey, S. Creager, C. Creutz, C.R. Kagan, P. V. Kamat, M.
Lieberman, S. Lindsay, R. a. Marcus, R.M. Metzger, M.E. Michel-Beyerle, J.R. Miller, M.D.
Newton, D.R. Rolison, O. Sankey, K.S. Schanze, J. Yardley, and X. Zhu, The Journal of
Physical Chemistry B 107, 6668 (2003).
SI.5 Analytical performances of the biosensor
The kinetic characterizations of electrochemical DNA sensors have been studied through
scan rate variation of the hybridization in the case of microfluidic cell and macro- cell.
Figure SI.5. CVs recorded after the hybridization at different scan; the top inset shows the
potential variation versus scan rate whereas the down inset shows the current variation with
the scan rate: (a) in the macro-cell and (b) in the microfluidic cell.
The variation in anodic potential (Epa) and cathodic potential (Epc) versus logarithm of the
scan rate (top insets) allows to calculate the rate of electron transfer ks following Laviron
model from the equation 5:
1 nvF 1

m RT k S
where n is the number of electrons transferred in redox reaction (n=1), v is the scan rate, m is
the dimensionless rate constant and R, T, F have their usual meanings.
Furthermore, the surface coverage could be calculated from the slopes of the linear plots of I
versus the scan rate and according to the following Randles-Sevcik equation:
Ip 
n 2 F 2 Av
4 RT
where n represents the number of electrons involved in reaction (one electron), A is the
surface area of the electrode (0.01 cm2 in microcell; 0.07 cm2 in macro-cell), T is temperature
(300 K),  (mol.cm-2) is the surface coverage. The surface coverage of the modified AuMWCNTs-Fc surface is estimated to be respectively 3×10-8 mol.cm-2 in the macro-cell and
0.45×10-8 mol.cm-2 in the micro-chamber (Table SI.5).
All this electrochemical parameters obtained is shown in the table below.
-08
MWCNTs-Fc-dsDNA
-2
-1
Γ (10 mol .cm )
Ks (s )
Macrocell
Microfluidic
cell
Macro- Microfluidic
cell
cell
1.3 ± 0.1 0.4 ± 0.04
1.2 ± 0.1 5.8 ± 1.2
Table SI.5. Electrochemical parameters obtained from electrodes of various modifications
Reference
5
E. Laviron, J. Electroanal. Chem. 101, 19 (1979).