sensor substrate functionalization for biosensing applications · 2012-03-23 · 50 nm steps and...

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: [email protected]

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.


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.


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.


-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.


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°.


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.


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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sensor substrate functionalization for biosensing applications · 2012-03-23 · 50 nm steps and...

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