Modified surface of titanium dioxide nanoparticles-based biosensor for DNA detection

Sh. Nadzirah, U. Hashim, and M. Rusop

Citation: AIP Conference Proceedings 1963, 020037 (2018); doi: 10.1063/1.5036883

View online: https://doi.org/10.1063/1.5036883
View Table of Contents: http://aip.scitation.org/toc/apc/1963/1
Published by the American Institute of Physics
 Modified Surface of Titanium Dioxide Nanoparticles-Based
Biosensor for DNA Detection
Sh.Nadzirah1, a), U. Hashim3, c) and M. Rusop1,2, b)
1
NANO-SciTech Centre, Institute of Science,
Universiti Teknologi MARA (UiTM), 40450 Shah Alam, Selangor, Malaysia
2
NANO-ElecTronic Centre, Faculty of Electrical Engineering,
Universiti Teknologi MARA (UiTM), 40450 Shah Alam, Selangor, Malaysia
3
Institute of Nano Electronic Engineering (INEE),
Universiti Malaysia Perlis (UniMAP), 01000 Kangar, Perlis, Malaysia
a)
shnadzirahsaa@gmail.com
b)
nanouitm@gmail.com
c)
uda@unimap.edu.my

Abstract. A new technique was used to develop a simple and selective picoammeter DNA biosensor for identification of
E. coli O157:H7. This biosensor was fabricated from titanium dioxide nanoparticles that was synthesized by sol-gel
method and spin-coated on silicon dioxide substrate via spinner. 3-Aminopropyl triethoxy silane (APTES) was used to
modify the surface of TiO2. Simple surface modification approach has been applied; which is single dropping of APTES
onto the TiO2 nanoparticles surface. Carboxyl modified probe DNA has been bind onto the surface of APTES/TiO2
without any amplifier element. Electrical signal has been used as the indicator to differentiate each step (surface
modification of TiO2 and probe DNA immobilization). The I–V measurements indicate extremely low current (pico-
ampere) flow through the device which is 2.8138E-10 A for pure TiO2 nanoparticles, 2.8124E-10 A after APTES
modification and 3.5949E-10 A after probe DNA immobilization.

INTRODUCTION
Escherichia coli (E. coli) is a species that can be categorized as a bacterium or germ that lives in the digestive
tracts of humans and animals. Most of E.coli is harmless and are important members of a healthy human intestinal
microbial community. However, there is an enterohemorrhagic E. coli (EHEC) which can lead to hemorrhagic
colitis [1]. Normally, this bacterium can get into raw meat, fruits, vegetables, milk and dairy products [2]. Other than
that, people can become infected when consuming contaminated water supply has not been properly treated with
chlorine. For prevention, it is important to pasteurize dairy products and cook meat more than 71 °C in order to
destroy the bacterium. E.coli is one of the pathogen which may cause foodborne disease and E.coli O157:H7 is the
major causes of illness and death [3]. Foodborne illnesses are diseases created by pathogens or environmental toxins
that enter the body through the consumption of food or water and can be infectious in nature [4]. Salmonella,
Campylobacter, Listeria, Escherichia coli, Vibrio cholera can be categorized as the major microorganisms involved
in these diseases.
Hence, several steps have been approached recently to control this issue such as check the food healthiness as
through good agricultural and manufacturing practices aspects [5]–[7], hazard analysis in critical control point
(HACCP) [8], [9] and food barcode reader devices to determine the expiry date of the products but it is not enough.
Thus, the development of new detection methods today that are able to detect the E.coli O157:H7 DNA in real time,
ultra high sensitivity and reproducibility are a big importance to prevent and identify problems related to health and
safety.

8th International Conference on Nanoscience and Nanotechnology 2017 (NANO-SciTech 2017)

AIP Conf. Proc. 1963, 020037-1–020037-5; https://doi.org/10.1063/1.5036883
Published by AIP Publishing. 978-0-7354-1668-0/$30.00

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 Metal oxide nanoparticles possess unique features as compared to the equivalent larger-scale materials [10]. For
biosensor applications, it is often necessary to functionalize the surface of metal oxide. Naturally, surface
modification starts with an existing surface and yields a new surface with possibly new properties, the area of
surface modification takes place between the particle synthesis and final possible applications [11]. The E. coli
O157:H7 DNA can only be immobilized onto the surface of TiO2 nanoparticles after chemical modification of the
TiO2 surface which is known as silanization has been. Generally, silanization is a process of covering the alkoxy
groups of TiO2 surface with APTES which has the silanol groups and form alkoxysilane compounds. Combination
of both the alkoxy and silanol groups generates the formation of siloxane linkages on the surface of TiO2 covalently
[12]. However, the reliability and consistency of the silanized surface have been issued. These are affected by the
solvent used for dilution purpose.
In this study, the development of TiO2-based biosensor for the detection of single-stranded E. coli O157:H7
DNA is described. In order to bind the organic and inorganic materials, the surface of inorganic TiO2 nanoparticles
was modified with NH2 linker using 3-Aminopropyl triethoxy silane (APTES). The formed amine group was
capable to covalently bind with the single-stranded probe DNA of E.coli O157:H7. The functionalization process
was completed with a simple drop of APTES onto the surface of TiO2, followed by a single drop of probe DNA.
Immobilization of probe DNA on the modified surface of TiO2 has successfully proved that the silanization process
is acceptable.

METHODOLOGY

TiO2 nanoparticles were synthesized using sol-gel spin-coating technique; as mentioned in the previous study
[13]. Electrodes were fabricated by depositing aluminum (Al) metal using thermal evaporator. The Al was then
fabricated using a conventional photolithography process. A pair of electrodes of 1.3 mm2 in size was fabricated on
the SiO2/Si substrate for the electrical measurement purpose. Figure 1 shows the design of the fabricated samples.
Then the electrical characterization was carried out using Kiethly6487 picoammeter interface with Labtracer2.0
software.
All the oligonucleotides used in sensor development were purchased from AITbiotech Pte. Ltd.
(Singapore). An E. coli gene with ssDNA probe 5’-AAC GCC GAT ACC ATT-3’ acted as positive control to
characterize the sensor performance. Silanization process used 1.5 µl of APTES which are simply dropped onto the
active area and was let to dry in vacuum cabinet for 1 h. After the cleaning process using deionized (DI) water, the
transducer was dropped with 1.5 µl single strand probe DNA and it takes 1 h to immobilize. The electrical signal
was measured before silanization, after silanization and after immobilization processes.

FIGURE 1. Fabricated interdigitated electrodes

RESULT AND DISCUSSION

Silanization is a process of introducing APTES with the purpose to modify the surface of TiO2 thus increases the
sensitivity and selectivity of the device towards E. coli DNA detection. TiO2 can be categorized as an inorganic
material; while DNA oligonucleotide is an organic material. It is impossible to directly bind the organic and
inorganic materials together. Hence, APTES was introduced on the surface of this metal oxide to overcome the
problem. The purpose of silanization process is to form bonds across the interface between organic (probe DNA)

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and inorganic (TiO2) materials. This process is also known as surface-modification process. The surface of TiO2
which has hydroxyl group (-OH) was covalently binded with APTES and formed –Si-O-Si- bond (siloxane bond) on
it.
To analyze the structural variations with varying contents (TiO2, APTES and DNA), the samples were first
characterized by using FTIR spectra. Figure 2 (a) displays the FT-IR spectra of pristine TiO2 before surface
modification. The IR band of lattice TiO2 can be obviously seen at 680 cm-1 which was attributed to the symmetric
stretching vibration of Ti-O-Ti bonds [14]. The band in the region of 800 cm-1 corresponded to the vibration of Ti-O
of the TiO2 nanoparticles [15]. The band of H-OH stretching vibration of physisorbed water can be seen at 1630 cm-
1
. The existence of APTES on the TiO2 surface can be obviously seen in Figure 2 (b). The IR band after surface
modification of TiO2 showed the asymmetric stretching vibration of the Si-O-Si bond at at 1130 cm-1 and 1040 cm-1
is due to the stretch vibration of Si-O-Si [16], [17]. These peaks suggest that APTES was successfully deposited on
the TiO2 surface. The 1562 cm-1 and 3389 cm-1 are assigned to the asymmetric and symmetric stretching modes of
NH2 (amine group). The hydrogen bond is strongly bonded to the silanol groups [17]. One may also point out a
stretching mode of CH2 at 2932 cm-1. Figure 2 (c) shows the signals of DNA backbone and phosphate groups of
nucleic acids which is at the region of 1200–800 cm−1. The peaks in the 1630-1697 cm-1 region are due to amide
bands of the protein in the cell [18].

(a) (b)

(c)

FIGURE 2. FTIR images of (a) bare TiO2 thin film; (b) modified surface of TiO2 thin film with APTES; and (c)
Immobilized TiO2 thin film with ssDNA of E. coli O157:H7

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 Figure 3 shows the current-to-voltage (I-V) curves of the TiO2 film after some modification and immobilization
processes. At 0.5V, the real current for bare TiO2 nanoparticles was 2.8137E-10 A and it decreased to 2.8124E-10 A
after APTES modification on the nanoparticles. However, after probe immobilization, the current increased greatly
to 3.5949E-10 A. During APTES modification, the particles diameter was covered with a layer of activated
aminosilane, thus reduced the diameter of particles which result in the decrement of the effective cross-section of the
area [19]. When the ion mobility through the particles was low, the current flow through the device reduces.

FIGURE 3. I-V curves of TiO2 nanoparticles for biosensor fabrication

After immobilization, the output current drastically increased. It can be explained as a consequence of the
increase in surface charge density from the negatively charged DNA. Binding of charged analytes to the particles
surface in this case affects the ion concentration on the particles [9].

CONCLUSION

In this paper, a simple surface modification approach has been introduced on the surface of TiO2 for detection of
E. coli O157H7 DNA. The synergistic effects of both TiO2 nanoparticles and APTES generated the output current
response due to the formation of new functional group (siloxane). Small volume of APTES has been introduced (1.5
μL). The output current response was measured after 1h of surface modification. This novel and rapid silanization
approach successfully binds the modified carboxyl probe DNA of E. coli O157:H7 with the siloxane. Each
interaction was observed by the changes of the output current response. This phenomenon indicates that this novel
surface modification approach showed the advantages such as simple preparation procedure, fast response, wide
dynamic detection range, and low volume consumption.

ACKNOWLEDGMENTS

This work is financially supported by LRGS grant (600-RMI/LRGS 5/3 (3/2013)), Institute of Research
Management & Innovation (IRMI), Universiti Teknologi MARA (UiTM), Research Chair for Biomedical
Applications of Nanomaterials, King Saud University and Ministry of Education Malaysia.

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