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Impedance spectroscopy measurements of a label free
functionalized silicon sensor
Ricardo M Trujillo1,4
, Anna Cattani-Scholz3, Achyut Bora
2, Vedran Vandalo
2
Rossana Madrid1 and Marc Tornow
2.
1National University of Tucumán, Av. Independencia 1900, Tucumán, Argentina.
2Institute of Semiconductor Technology, Technical University Braunschweig, Hans-
Sommer-Strasse 66, 38106 Braunschweig,Germany 3 Walter Schottky Institute, Technical University Munich, Am Coulombwall, 85748,
Germany
Abstract. There is not current commercial methods that allows to measure in real time
biological events or quantify molecules. The main disadvantage of present methods is that
samples must be labeled before the measurements.
This work describes a possibility of label-free biosensing based on crystalline silicon
functionalized with organic molecules. In order to get a response to changes on the sensor´s
surface a special oxidation technique is needed. This technique allows to obtain
uncontaminated ultra-thin layers and a very good quality of self-assembled monolayers
(SAMs).
Electrochemical impedance spectroscopy was used to analyze two particular cases of
electrode functionalization: 1)protein bonding and 2) non-specific adsorption of λ-DNA
over a self-assembled monolayer of molecules with different ending groups.
1. Introduction
A biosensor is a device designed to detect certain specimens of the organic world such as DNA
chains, proteins, antigen-antibody or even microorganism.
There are several types of biosensors. We may find: 1) nano-gold particles (AuNPs), which can
be used for improving biocompatibility or for the enhancement of biomolecule loading. 2) Nano
wires (NW), used as a FET sensor or as a template for bio-marking detection. 3) Nano pores, which
are employed for translocations studies.
Among current label free sensors we find many different types: optical sensor in the case of
surface Plasmon resonance (SPR), piezoelectric sensor e.g. Quartz crystal microbalance (QCM),
sensors based on electrical changes e.g. IgFet or capacitive where the dielectric constant changes
with variations in the solution.
In this paper we analyze the possibility of sensing changes on the functionalized surface when
the target molecule approaches or when a binding event occur. Functionalization of the surface was
done by Tethering aggregation and growth (TBAG) for C10-OH and C17-CH3 molecules. Biotin
and Streptavidin were deposited by a different chemical method each one.
Electrochemical impedance spectroscopy is a useful method to characterize the interaction
between molecules and the sensor surface. In this work, we choose this technique to analyze
4 To whom any correspondence should be addressed at: [email protected].
XVIII Congreso Argentino de Bioingeniería SABI 2011 - VII Jornadas de Ingeniería Clínica Mar del Plata, 28 al 30 de septiembre de 2011
changes in substrate with and without functionalization, binding between biotin-Streptavidin and
nonspecific adsorption of Lambda DNA chains. [1-5]
2. Materials and methods
2.1. Impedance background
A small ac signal is applied to analyze the properties of the modified electrodes’ surface. The
impedance value is obtained from the relationship between the applied voltage and the current, and
it can be expressed as the sum of real and the imaginary parts.
Z= Zre+Zimg (1)
In general, the measured value is the result of the sum of the contribution of all resistances and
capacitances of the electrolyte cell and electrodes. Information can be extracted by comparing
measured data to a fitting circuit, although there is not an exact correspondence or a unique physical
circuit that models the phenomena. In addition, components values will change according to the
frequency range studied.
It can be assumed that capacitance values correspond to a parallel capacitor with:
(2)
Where is the isolator permittivity, 0, is the vacuum permittivity, A is the area under study and t is
the thickness of the isolator layer. The measure capacitance value is:
(3)
Where Ct is the total capacitance, Cdl models the electrical double layer and Cmod is the capacitance
resulting from the modification on the samples’ surface.
Gouy-Chapman-Stern model indicates that the electrical double layer has three zones: 1) the
inner Helmholtz zone, 2) the outer Helmholtz zone and 3) the diffuse zone. Each one of these zones
has an associated capacitance in series with the oxide layer capacitance. According to Fung et al.
typical values for diffusion zone capacitance is in the order of F/cm2, CIH (inner Helmholtz
capacitance) is around 140 F/cm2, and COH (outer Helmholtz capacitance) is ca. 20 F/cm
2. These
capacitances play a minor role in impedance sensors since they are much greater than the oxide layer
or the functionalization layer capacitances.
A capacitive-like element can be used to model the interface; this element is the constant phase
element (CPE). It represents the inhomogeneous surface of the electrode and it is described by the
following equation:
(4)
Where C is the ideal capacitance, , is the angular frequency and n is an empirical constant without
a physical base. When the n value is bigger than 0.85, the CPE can be considered as a capacitance
and for n> 0.9, C becomes a pure capacitance.
The interface has another element, the charge transfer resistance, Rtc. This resistance is the result
of the transfer resistance of the electrode without modification, Rto plus the resistance due to the
surface modification, Rmod. The electrolyte solution has also a resistance, Rsol, this is a consequence of
the ions finite conductance.
In absence of redox reactions, only non-faradic current is present. In this case the circuit’s
variable is Ct. Therefore we can model the interface as the following picture [6-10]:
XVIII Congreso Argentino de Bioingeniería SABI 2011 - VII Jornadas de Ingeniería Clínica Mar del Plata, 28 al 30 de septiembre de 2011
Figure 1. Equivalent circuit.
2.2. Electrochemical cell
In this paper we use a monovalent buffer as the measurement medium. The cell has three
electrodes: working, reference and counter electrode. The working electrode is the sample to be
measured, reference is a standard Ag/AgCl electrode (Microelectrodes Inc) and a 1 mm in diameter
platinum wire (99,99% purity, Umicore) is the counter electrode. The electrical potential of the cell
is:
(5)
Where , is the solutions’ potential; the interfase potential is . The drop in
potential within solution is . Thus:
(6)
The electrochemical cell used has two main parts: a copper base that provides electrical contact
between the working electrode and the measurement equipment. The solution compartment was
made of acrylic, which is attached to the base with two metallic screws. Between the sample and the
acrylic is placed an O-ring to avoid buffer leakage.
All measurements were carried on inside a Faraday cage and the equipment used was Ivium
CompactStat (Ivium Technologies, Netherlands).
In the experiments, frequency range was from 20 Hz to 100 kHz, ac signal was a sin wave with
10 to 25 mV rms. Buffer and DNA solution were injected by a 20 ml syringe (Braun, Germany),
using for this purpose Luer-lock connectors and silicone tubes. Before the measurements started,
the buffer was taken from refrigerator. Ambient temperature inside the laboratory was ca. 22°C
controlled by an air conditioner. [7-11]
Samples were functionalized with C10-OH and Biotin + Biotin-Streptavidin at the Institute for
Semiconductor technology (IHT, TU Braunschweig, Germany) chemical laboratory. Silicon samples
with C17-CH3 on top were functionalized at the Walter Schottky laboratory in Munich, Germany.
3. Results and discussion
3.1. SiO2 + SAMs 2TBAG
These samples were functionalized twice with C10-OH over a ca. 2 nm SiO2 layer. The buffer used
was 20 mM Tris HCL. Measurements were done with two different Vbias, 0 V and 20 mV. Picture 2
shows the module and the phase of the impedance versus frequency in logarithmic scale. Figure 3
shows a Nyquist (Z imaginary vs. Z real) plot where difference can be noticed easily.
XVIII Congreso Argentino de Bioingeniería SABI 2011 - VII Jornadas de Ingeniería Clínica Mar del Plata, 28 al 30 de septiembre de 2011
101
102
103
104
105
1k
10k
0 V SAMs
20 mV SAMs
0 V SiO2
20 mV SiO2
10
Lo
g |Z
| [O
hm
]
freq. /Hz
20 mM Tris HCl
HD b8
Figure 2 Module of Z vs. frequency plot of the
reference (SiO2) vs SAMs at two different Vbias.
3,0k 6,0k 9,0k
-40,0k
-20,0k
0,0
0 V SAMs
20 mV SAMs
0 V SiO2
20 mV SiO2
Z2
/o
hm
Z1 /ohm
20 mM Tris HCl
HD b8
Figure 3. Nyquist plot of the reference (SiO2) vs SAMs
at two different Vbias.
Improvement in the quality of the surface functionalization allowed a bigger difference between
the two surfaces, therefore this factor could be taken as an indicator of the deposition quality.
3.2. Biotin vs Biotin-Streptavidin
Biotin was deposited on samples with C10-OH SAMs. One sample was left to serve as reference and
in the other one Streptavidin was deposited. Measurements were done on 20 mM Tris HCl buffer at
different Vbias. In this paper we show only the results for Vbias= 0V in the following figures:
XVIII Congreso Argentino de Bioingeniería SABI 2011 - VII Jornadas de Ingeniería Clínica Mar del Plata, 28 al 30 de septiembre de 2011
101
102
103
104
105
1k
10k
Biotin
Streptavidin-Biotin
10
Lo
g |Z
| [O
hm
]10Log f [Hz]
0 V
Figure 4. Module of Z vs. frequency of the reference
(biotin) and Biotin + Streptavidin.
1,2k 1,8k 2,4k
-20k
-10k
0
Biotin
Streptavidin-Biotin
Z2
/o
hm
Z1 /ohm
0 V
Figure 5. Nyquist plot of the reference (biotin) and
Biotin + Streptavidin.
Biotin-Streptavidin bond is one of the strongest unions between proteins. In order to study the
substrate’s behavior we used this particular feature. Best detection was achieved at Vbias= 0V,
although a bigger change was expected. Since we only had one sample to make the experiment is
very possible that deposition was not optimal, thus resulting in smaller impedance changes.
3.3. C17-CH3 vs C17-CH3 + DNA
These measurements were carried out at Vbias= -25 mV. A negative voltage was chosen since DNA
has a negative net charge; therefore it was intended to enhance the approach of the chain to the
surface. Reference was taken to be the substrate where, a buffer similar to the DNA one was injected
and measured. The injections of buffer with and without DNA were 200, 500, 700 and 900 l.
XVIII Congreso Argentino de Bioingeniería SABI 2011 - VII Jornadas de Ingeniería Clínica Mar del Plata, 28 al 30 de septiembre de 2011
101
102
103
104
105
1k
10k
20 mM Ref vs CH3+DNA -25 mV
|Z| vs f
Ref 200 micrl
Ref 500 micrl
Ref 700 micrl
Ref 900 micrl
DNA 200 micrl
DNA 500 micrl
DNA 700 micrl
DNA 900 micrl
10
Lo
g |Z
| [O
hm
]
10Log f [Hz]
Figure 6. Module of Z vs. frequency in logarithmic
scale at different buffer concentrations.
1k 2k 3k 4k 5k
-35,0k
-30,0k
-25,0k
-20,0k
-15,0k
-10,0k
-5,0k
0,0
5,0k
Ref 200 micrl
Ref 500 micrl
Ref 700 micrl
Ref 900 micrl
DNA 200 micrl
DNA 500 micrl
DNA 700 micrl
DNA 900 micrl
Z2
/o
hm
Z1 /ohm
-25 mV Ref vs CH3-DNA
Figure 7. Nyquist plot of Reference vs. DNA at
different concentrations.
The module and phase of the impedance did not show great changes as expected for these
molecules where the 200 l plot is an exception. These molecules have an end group which is
hydrophobic and they also have a negative net charge. For these reasons it was not expected big
changes between reference sample and DNA. It is possible that the difference observed for the 200
l plot is due to the injection of the buffer where DNA is dissolved, modifying the electrolyte
conductivity of the solution.
3.4. C10-OH vs C10-OH + DNA
In these experiments, the measurement protocol was the same one that for the C17-CH3. In this case,
two different bias were used: -10 mV and -15 mV.
101
102
103
104
105
1k
10k
-10 mV SAMs
-15 mV SAMs
-10 mV DNA
-15 mV DNA
10
Lo
g |Z
| [H
z]
10Log f [Hz]
200 micrl Ref vs OH-DNA
|Z| vs f
Figure 8. Module of Z vs. frequency.
XVIII Congreso Argentino de Bioingeniería SABI 2011 - VII Jornadas de Ingeniería Clínica Mar del Plata, 28 al 30 de septiembre de 2011
1,0k 1,5k 2,0k 2,5k 3,0k
-60k
-50k
-40k
-30k
-20k
-10k
0
Ref 500 micrl
Ref 700 micrl
Ref 900 micrl
DNA 500 micrl
DNA 700 micrl
DNA 900 micrl
Z2
/o
hm
Z1 /ohm
-10 mV Ref vs OH-DNA
Figure 9. Nyquist plot for different buffer
injections, with and without DNA (reference).
Molecules with OH end group are less hydrophobic than the CH3 end group molecules. In
addition they have a positive net charge. These differences should enhance DNA adsorption to the
surface but changes were less than expected. Most measurements were noisy at low frequencies,
which is most probably due to buffer leakage and low surface ratio between working and counter
electrodes, having a much influence in measurements.
The following tables show the mean value and the standard deviation for each impedance
experiment. These values were obtained comparing impedance data with a fitting circuit. Such
analysis was done in Iviumsoft using a resistance with a CPE in series.
3.5. SiO2 (2nm) vs. 2TBAG SAMs (C10-OH)
Table 1.
Vbias= 0V
Ref SAMs
R (Ohm) C (nF) R (kOhm) C (nF)
Media StadDev Media StadDev Media StadDev Media StadDev
952,7 0,8 45,536 0,567 1,127 0,008 251,1 1,679
3.6. Biotin vs Biotin-Streptavidin
Table 2.
Vbias= 0V
Ref Bio&Strept.
R (kOhm) C (nF) R (kOhm) C (nF)
Media StadDev Media StadDev Media StadDev Media StadDev
1,106 0,008 252,3 0,6 1,102 0,009 334,5 1,0
XVIII Congreso Argentino de Bioingeniería SABI 2011 - VII Jornadas de Ingeniería Clínica Mar del Plata, 28 al 30 de septiembre de 2011
3.7. SAMs (C17-CH3) vs. SAMs (C17-CH3) + DNA
Table 3.
Vbias= -25 mV
Ref DNA
Injection R (kOhm) C (nF) R (kOhm) C (nF)
l Media StadDev Media StadDev Media StadDev Media StadDev
200 1,082 0,007 112,1 0,6 1,131 0,003 117,9 0,4
500 1,140 0,003 101,5 0,8 1,130 0,003 112,7 0,6
700 1,187 0,004 103,9 0,6 1,124 0,003 116,9 0,3
900 1,230 0,004 105,1 0,6 1,189 0,004 119,6 0,4
3.8. SAMs 2TBAG (C10-OH) vs. SAMs 2TBAG (C10-OH) + DNA
Table 4.
Vbias= -10mV
Ref DNA
Injection R (kOhm) C (nF) R (kOhm) C (nF)
l Media StadDev Media StadDev Media StadDev Media StadDev
500 1,133 0,383 305,2 1,7 1,215 0,434 227,1 1,5
700 1,125 0,559 296,5 0,8 1,245 0,786 232,9 1,1
900 1,146 0,525 294,1 1,1 1,251 0,663 226,8 0,8
Statistical analysis shows, for all experiments, that the mean (R and C) values are statistically
different for a 95% confident interval.
4. Conclusion
Further studies regarding DNA and other molecules should be carried out in order to enhance the
measurement system. The main goal is to obtain a suitable substrate, with surface functionalization,
to develop bigger projects like NanoLab-on-a-chip. In this work we obtain a statistical difference
between the impedance measurements of the different molecules.
Future work should focus on depositing other molecules on top the functionalized surface such
as a heterobifunctional linker or a peptidic nucleic acid (PNA) which is analogue to DNA and
serves as a probe molecule to bind it. For all this it is important to improve surface functionalization
processes.
Acknowledgement I am very thankful to Prof. Dr. Marc Tornow, Dr. Anna Cattani-Scholz, Dr. Achyut Bora, MSc.
Vedran Vandalo, MSc. Anshuma Patak and Dipl.-Ing. Muhammed Ihab Schukfeh for their helpful
knowledge and experimental support. This work was supported by the Institut für Halbleiter
Technik (IHT), TU Braunschweig. I am grateful to the Deutcher Akademischer Austausch Dienst
(DAAD) for granting me with a scholarship and Dr. Rossana Madrid for a critical reading of the
manuscript.
XVIII Congreso Argentino de Bioingeniería SABI 2011 - VII Jornadas de Ingeniería Clínica Mar del Plata, 28 al 30 de septiembre de 2011
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XVIII Congreso Argentino de Bioingeniería SABI 2011 - VII Jornadas de Ingeniería Clínica Mar del Plata, 28 al 30 de septiembre de 2011