sensor substrate functionalization for biosensing applications · 2012-03-23 · 50 nm steps and...
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Sensor substrate functionalization for biosensing applications
Ricardo M Trujillo1,4
, Anna Cattani-Scholz3, Achyut Bora
2 Anshuma Patak
2
Rossana Madrid1 and Marc Tornow
2.
1National University of Tucumán, Av. Independencia 1800, 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..The main disadvantage of the present sensing methods is that samples must be
labeled previous the measurements. In some cases, to obtain a reliable result, it is necessary to
use considerable amount of analyte, or it is not possible to label it, making difficult to carry out
these measurements.
This paper describes the possibility of label-free biosensing based on crystalline silicon
functionalized with organic molecules. This work started with the improvement of the p type
silicon substrate by doping its back side with Boron. Several techniques were used to prepare
the semiconductor, among them we find optic lithography, oxidation, doping and metallization.
In order to get a response, it is necessary to prepare the sensor´s surface with a special
oxidation technique that allows uncontaminated ultra-thin layers. Very good ohmic contact
between sample and electrode is also needed. For this reason, extra Boron dopands were used
in addition of the metal evaporated to create the contact.
Key words: Semiconductor biosensors, “label-free”, functionalization with alkyl-phosphonate
monolayers.
1. Introduction
Silicon-based technology provides a very good combination of stability, biocompatibility and
miniaturization. The combination of solid state technology with a biological environment allows
greater efficiency, compact systems and better detection capabilities. This will be useful for patients in
difficult locations, as high mountains, or for the early detection of chemical or biological agents in
warfare.
Current commercial sensors require some kind of label in order to record a signal. The most known
example of these devices is the DNA-chip, which uses a fluorescent label to analyze the samples.
Detection using label-free devices reduces greatly time and cost since there is no need to prepare the
sample for its detection i.e. labeling steps. Within this kind of sensors we find Quartz crystal
microbalance (QCM), Surface Plasmon Resonance (SPR), and sensors based on changes of theirs
electrical properties e.g. IgFet, impedance spectroscopy detection, or DNA-nanopores which measure
a signal resulting from translocation event throw the pore. In this later case it is important to prevent
DNA nonspecific adsorption at the sensor walls.
In this work we present the fabrication of a sensor, for label free impedance studies in biosensing
applications. The starting material was p type silicon, where different processes were carried on. First
we modified back contact by introducing dopands and making metal evaporation. On the sample´s
4 To whom any correspondence should be addressed at: rmatias.trujillo@gmail.com
XVIII Congreso Argentino de Bioingeniería SABI 2011 - VII Jornadas de Ingeniería Clínica Mar del Plata, 28 al 30 de septiembre de 2011
front side ultra-thin oxide layer were grown and it was also functionalized with different organic
molecules. [1-5]
2. Materials and methods
2.1. Substrate preparation
Two different planar p type silicon electrodes were considered for the studies, highly and low doped,
that is 1018
1/cm3 and 10
16 1/cm
3 respectively. In order to obtain a suitable detection signal, the
substrate must be modified. This process involves several steps, the first one is to create an Ohmic
contact between the working electrode and the measurement equipment. This was achieved by doping
the back side with boron impurities and evaporating Cr/Au (35 nm/500 nm). Another step in the
substrate modification was the functionalization of the front side, this includes ultra-thin thermal oxide
growth and deposition of Self assembled monolayers (SAMs) using alkylphosphonate molecules with
different end groups and back bones’ longitudes. For this purpose standard Tethering by aggregation
and growth (TBAG) deposition was used, in the case of biotin a different method was used. We used
several analytical methods to characterize the surface and the ohmic contacts, such as transmission
line, ellipsometry, contact angle, X-ray photoelectron spectroscopy (XPS) and Atomic force
Microscopy (AFM). This work concerns both detection and isolation of the substrate as part of nano-
pore DNA translocation project at the Univerität Braunschweig. In the later case DNA must not be
detected by the substrate.
Starting material was (100) oriented silicon wafer (CrysTec). Most experiments were performed
with p-type Si (15-25 mΩ cm) and in some cases were compared with p-type Si low doped (1-40 Ω
cm). All wafers were cut first in 27x27 mm to start processing and then cut in 9x9 mm to measure.
Wafers were cut by scribing it with diamond tip and manually pressing to separate the samples.
Previously start, all samples were cleaned using standard Piranha solution for 5 minutes at 75 °C,
heavily rinsed with distilled water and dried with N2 blow. High doped wafer were purchased with 100
nm SiO2 layer while low doped wafer were purchased without it. This layer was advantageous since
provides protection to the front side from boron diffusion. Protection layer was created in low doped
wafer by thermal growth at 1150 °C in 3.5 l/min flow of dry O2, after this oxidation, annealing was
made at the same temperature in 3.5 l/m flow of N2.
Extra doping of the back side was achieved by introducing boron impurities (Boron film 100,
Emulsitone CO., USA) using thermal method. Boron solution was spinned over the back side inside a
clean room. This solution contains a water-based solvent which was removed evaporating it in an oven
at 200 °C for 30 minutes. Finally, diffusion was made at 1050 °C for 60 minutes. To avoid any
thermal stress, samples were pushed forward and pulled back with a mechanic arm at a constant time
rate ca. 5 minutes each way. Before and after doping, the substrate was immersed in HF solution (5%
in water, VSLI selectipur line, BASF) for 4 and 8 minutes respectively. Bathing samples in HF is
necessary to remove all SiO2, thin layers were found to act as a mask preventing diffusion. Immersing
the samples in HF after thermal diffusion allows removing the borosilicate layer formed in the back
side. Protection from the boron solution to the anterior surface was made by spinning photoresist and
covering it with thermal paper.
Metal contact was made by standard metal evaporation method. Chromium adhesion layer was
chosen over Titanium since Cr is not etched by HF acid. In order to protect the front side, spinning
photoresist was necessary.
Ultra-thin SiO2 was investigated using naturally grow SiO2 and thermal growth. Natural silicon
oxide was grown over night inside clean room at room temperature. Before thermal growth, HF
immersion is needed to remove the natural SiO2, this creates a H-terminated surface, this H layer is
known to last for ca. 30 minutes. Thermal SiO2 was achieved at 600 °C and 2 l/min dry O2 flow. After
oxidation, annealing was performed by using dry N2 in order to improve SiO2 mechanical and
electrical properties. Immediately after this process, photoresist is spinned to protect the oxide layer.
XVIII Congreso Argentino de Bioingeniería SABI 2011 - VII Jornadas de Ingeniería Clínica Mar del Plata, 28 al 30 de septiembre de 2011
Figure 1. Schematic illustration of the diffusion process.
[6-9]
2.2. Contact evaluation (Transmission line method)
This assay was made on the sample´s back side to evaluate the quality of the ohmic contact and developed
inside clean room´s yellow part to avoid contamination and exposure of the materials to external UV light
such as fluorescent lamps or sun light.
Lines were built by UV-lithography technique. Negative photoresist (ma-N 1420, Microresist
Tech., Germany) was spinned coated on the surface, backed on hot plate at 160 °C for 60s, and
exposed to UV light in the mask aligner at 8mW for 70 s using a line patterned glass mask. Samples
were developed using ma-D 533 S (Microresist Tech., Germany) for 80 s, rinsed in DI water and blow
dried with N2. To assure the substrate is cleaned, oxygen plasma was applied at 200 mW for 1 minute.
Immediately before metal deposition, samples were immersed in HF solution to remove all SiO2.
Transmission line samples were immersed in acetone over night for lift-off, then ultrasonicated in
clean acetone, and finally rinsed with Isopropanol and dried with N2 blow.
Transmission lines allows to measure resistance between the metal contact and the semiconductor
substrate. The obtained value is the sum of the contact resistance, Rc, of two consecutive lines and the
resistance of the substrate between them.
(1)
Where is the material resistivity, A is area and l the separation between consecutive contacts.
XVIII Congreso Argentino de Bioingeniería SABI 2011 - VII Jornadas de Ingeniería Clínica Mar del Plata, 28 al 30 de septiembre de 2011
2.3. Molecules deposition
Samples were cleaned in a H2O, NH4OH and H2O2 (5:1:1 in volume) solution at 80 °C for 10 minutes
to create OH-terminated samples. These samples were functionalized with two different molecules,
C10-OH (11-hydroxyundecylphosphonate) and C17-CH3. Deposition was made by TBAG process.
TBAG deposition process is made twice to obtain better surface coverage. [4]
Using a different chemical procedure samples were also cover with biotin and Streptavidin
molecules for binding experiments.
2.4. Contact angle
A 5 l drop is deposited by a microsyringe on the sample´s surface. The wetting angle between the
surface and the drop is measured using a CCD camera and special software. Surface energy can be
related to the contact angle according the following equation:
(2)
Where is the free energy of the solid surface in contact with air; , is the free energy of the solid-
liquid face and , is the free energy of the liquid in contact with air.
3. Results and discussion
3.1. Thermal diffusion
Concentration profile was measure with “Wafer Profiler CVP 21”. Samples are etched by UV light in
50 nm steps and stops at 6 m, electrolyte is injected and a Capacitive vs. Voltage (C-V) measure is
taken. Doping concentration is taken from the slope of 1/C2. Results are shown in the following
graphic.
0 2 4 6
1018
1019
Na (
cm
-3)
Depth (m)
Figure 2. Boron concentration profile on samples’
back side.
The obtained concentration profile showed a behavior according to the given the conditions under
which the diffusion was made. Dopands penetration in the substrate was less than expected. These
differences have several causes, this technique requires great manipulation and training from operator,
the substrate must be clean of SiO2 since this surface acts as a mask. One of the process’ steps is the
evaporation of the water-based solvent, the oven for this purpose lacks of modified atmosphere so an
oxide layer grew. This layer was thin enough to allow a limited range of penetration.
XVIII Congreso Argentino de Bioingeniería SABI 2011 - VII Jornadas de Ingeniería Clínica Mar del Plata, 28 al 30 de septiembre de 2011
-2 -1 0 1 2
-0,4
-0,3
-0,2
-0,1
0,0
0,1
0,2
0,3
0,4
Cu
rre
nt (A
)
Voltage (V)
400 micros
600 micros
800 micros
1000 micros
1200 micros
Low Doped bonded
B)
-1,5 -1,0 -0,5 0,0 0,5 1,0 1,5
-0,6
-0,4
-0,2
0,0
0,2
0,4
0,6
Cu
rre
nt (A
)
Voltage (V)
400 micros
600 micros
800 micros
1000 micros
1200 micros
Hi Doped bonded
A)
600 700 800 900 1000
2,288
2,290
2,292
2,294
2,296
2,298
2,300
2,302
2,304
2,306
Resistance
Linear Fit of Resistance
Re
sis
tan
ce
(O
hm
)
Longitud (micrometers)
A)
400 600 800 1000 1200
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
R vs L
fitting
Re
sis
tan
ce
(O
hm
)
Longitug (micrometers)B)
3.2. Transmission lines
These experiments were carried out inside a Faraday box at the electrical laboratory. Measurements
were taken with Keithley 2635 System Sourcemeter (LabTracer 2.0 software). This equipment applies
a voltage from -2V to +2V in 500 steps, records current and plots an I-V curve (figure 3). Data points
were acquire from each line at the different separations, 200, 400, 600, 800, 1000 and 1200 m. Each
resistance is fit to a line and the interception is the contact resistance (figure 4).
For this study two different methods were considered, probe station with needle electrodes and
chip-carrier with gold on gold bonding.
Figure 3. Transmission line measurements on bonded samples of A) High doped and B) low dopes
Figure 4. Contact resistance on A) high dopes and B) low doped silicon samples.
Rcontact= 140,0 ± 5,0 [Ω/cm2] for High doped silicon.
Rcontact= 320,0 ± 8,0 [Ω/cm2]
for low doped silicon.
In general these measurements were noisier than expected, none the less it was possible to obtain
the contact resistance value. Low dope substrate had a better linear fit than the high doped one but the
last one had a lower contact resistance. This indicates that diffusion was more uniform in low doped.
XVIII Congreso Argentino de Bioingeniería SABI 2011 - VII Jornadas de Ingeniería Clínica Mar del Plata, 28 al 30 de septiembre de 2011
Both substrates showed a straight line behavior so the main objective of this study was achieved.
Some data were neglected like the ones obtained for 200 and 1200 m. This was done due to problems
in the measurement set-up or to the sample itself like surface´s roughness.
3.3. Thickness measure by Ellipsometry
Measurement were taken with ELX-02 ellipsometer, equipped with HeNe laser (λ=632.8 nm) at an
incident angle of 70°. Oxide thickness was obtained by averaging measurements in five different
points for each sample, as shown in figure 6. The results are summarized in table 1.
Figure 6. Points of
measurement at
samples’ surface.
Table 1. Summary of the ellipsometry measurements’ results.
Temp. Time in O2 Time in N2 Typea
Sample Thickness StadDev Notes
°C min min nm
Amb. 9 Hourss - HD ON1 1,4723 0,0333 Ox. Native
Amb. over night - HD ON3 2,1056 0,1487 Ox. Native
600 5 10 HD B8-A 2,1811 0,0218 -
600 5 10 HD B9 2,1046 0,1034 no Dif
600 10 10 HD B9-c 4,5651 0,1805 no Dif
600 5 10 HD B10-C 2,1349 0,0886 Ref
1150 60 45 LD P3 101,4598 0,5618 Protection
600 5 10 LD B7-a 2,0949 0,0983 - aHD: high doped; LD: low doped
Three measurements were done in each of five points on the sample´s surface. The results shows
that samples with native oxide (ON 1-3) showed a fast initial growth followed by stabilization since
oxide itself acts as a mask for further oxidation. This is a great advantage for ultra-thin layers, main
disadvantage of this process is contamination and poor electrical properties compare to the thermal
oxide. While growing ultra-thin layers is much complicated using thermal process, it allowed a better
quality. Controlled atmosphere with dry O2 gas reduced contamination problems and annealing with
N2 improved both mechanical and electrical features of the oxide layer.
3.4. Contact angle measurements
These measurements were taken from 1 or 2 samples of 9x9 mm per batch. In every sample 4 angles
were measured at different locations. A DI water drop was deposited with a microsyringe, recording
the angle immediately (see table 2). The measured mean value have a standard deviation of 6°.
XVIII Congreso Argentino de Bioingeniería SABI 2011 - VII Jornadas de Ingeniería Clínica Mar del Plata, 28 al 30 de septiembre de 2011
Table 2. Measured angles
on the different surfaces.
Surface Angle
Oxide ca. 20°
-OHa
71,4 °
-CH3a
112,8°
Biotin 73,4° a Molecules’ end-group.
This measurement is a very fast way to analyze surface feature of a given sample, although
obtained results from this technique must be taken with precaution. Different angles might result from
variation in ambient condition such as temperature, pressure or humidity. Reference line, from where
the angle is measure, is established by the researcher, thus it changes in every measurement. Other
problems like contamination on the sample also can influence the measurement.
3.5. Atomic Force microscope measurements An AFM from Veeco instruments, working in sweeping mode, was used to study the surface. AFM
analysis was done on surface with C10-OH SAMs in substrates after two depositions. The studies
showed a surface coverage with a root mean square (rms) roughness of 0.3 nm in concordance with
Cattani et al. (2008) work. Samples with multilayers showed a roughness (rms) of 0.7 nm (see Fig. 7 A
and B)
Figure 7. AFM pictures of 11-hydroxyundecylphosphonate on SI/SiO2 surface.
A) smooth surface after two depositions. B) after two depositions where white
spots are considered to be multilayers.
The first samples analyzed showed multilayers (Fig 7 B). Figure 7 shows AFM analysis enabled
the evaluation of the complete fabrication process. The main problem founded was the contamination
along all the development of the sample, such as metal on surface from evaporation step or humidity
in glassware during deposition step.
XVIII Congreso Argentino de Bioingeniería SABI 2011 - VII Jornadas de Ingeniería Clínica Mar del Plata, 28 al 30 de septiembre de 2011
3.6. XPS
Composition of the surface was studied by X-ray photoelectron spectroscopy in TU München (Dr.
Anna Cattani-Sholz). In samples with C10-OH phosphorus P2p doublet was detected indicating the
presence of molecules. In samples with Biotin and Streptavidin a double peak for C1s and a single
peak for N1s were found indicating the successful deposition of biotin and the union between biotin-
streptavidin (data not shown).
4. Conclusion Results presented here show a fairly easy, cheap and fast fabrication of a suitable substrate for a label
free sensor type.
In this work we achieved a satisfactory thermal diffusion of dopands, enhance semiconductor-metal
ohmic contact, an ultra-thin SiO2 layer and the successful deposition of different molecules with good
surface coverage.
Experiments with silicon technologies allow integration of different sensors into a unique chip.
Future work will focus on the impedance analysis for DNA detection or reducing its nonspecific
adsorption. Impedance spectroscopy will allow studying the behavior of the functionalized sensor´s
surface.
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. 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.
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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
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