Impedance spectroscopy measurements of a label free ... · PDF fileNational University of...

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]:


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.


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:


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.


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.


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


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.


References

[1] Magdalena G. y Grzegorz W., An introduction to DNA chips: principles, technology,

applications and analysis 2001, Acta Bioquimica Polonica Vol. 48 n° 3.

[2] Vidotti M., Carvalhal R. F, Mendes R. K., Ferreira D. C. M. y Kubota L. T., Biosensors

based on Gold Nanostructures 2010, J. Braz. Chem. Soc., Vol. 00, No. 00, 1-18.

[3] Label-free amplified bioaffinity detection using terahertz wave technology 2004, Menikh A.,

Mickan S. P., Liu H., MacColl R., Zhang X., Biosensors and Bioelectronics 20 658–662.

[4] Cattani-Scholz A., Pedone D., Dubey M., Neppl S., Nickel B., Feulner P., Schwartz J.,

Abstreiter G. y Tornow M., Organophosphonate-Based PNAFunctionalization of Silicon

Nanowires for Label-Free DNA Detection 2008, ACSNANO VOL. 2 NO. 8 1653–1660.

[5] Yaling L., Samir M. I., Silicon-based novel Biosensing platforms at the micro and nano scale

2009, ECS Transactions The Electrochemical Society, 16 (15) 25-45.

[6] Orazem M. E. , Tribollet B. Electrochemical Impedance Spectroscopy 2008,

Wiley-Interscience,.

[7] Bard A. J., Faulkner L. R., Electrochemical Methods: Fundamentals and Applications 2000,

Wiley.

[8] Macdonald J. R., Impedance Spectroscopy, Annals of Biomedical Engineering 1992, Vol. 20,

pp. 289-305.

[9] Boddy P. J., Impedance Measurements at the semiconductor-electrolyte interface 1969,

Surface Science 13.

[10] BOUSSE L., BERGVELD P., On the impedance of the silicon dioxide/electrolyte interface

1983, J. Electroanal. Chem., 152 25-39

[11] Kovhcs I.K., . Horvai G, Possibilities of chemical sensing at the semiconductor/electrolyte.

Interface 1994, Sensors and Actuators B, 18-19 315-320.


Impedance spectroscopy measurements of a label free ... · PDF fileNational University of...

Documents

Transcript of Impedance spectroscopy measurements of a label free ... · PDF fileNational University of...