Babes -Bolyai University [627917]

Babes -Bolyai University
Faculty of Physics
Specialization Technological Physics

Master Thesis

DEVELOPMENT OF SERS SUBSTRATE
FOR DETECTION OF OCHRATOXIN A

Scientific advisor Romania
Prof. Univ. Dr. AȘTILEAN Simion
Scientific advisor France Graduate
SABĂU Alexandru Gheorghe
Pr. Dr. LAMY de la CHAPELLE Marc

CLUJ -NAPOCA
2017

DEVELOPMENT OF SERS SUBSTRATE
FOR DETECTION OF OCHRATOXIN A

Scientific advisor s Romania
Prof. Univ. Dr. AȘTILEAN Simion
CS I. Dr. Focșan Monica
Graduate
SABĂU Alexandru Gheorghe
Scientific advisors France
Pr. Dr. LAMY de la CHAPELLE Marc
Dr. Gillibert Raymond

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Contents

Thesis outline ………………………….. ………………………….. ………………………….. ………………………….. ……. 4
Chapter 1 Nanostructures – General Aspects – Literature ………………………….. ………………………… 6
1.1 Fabrication methods of nanostructures ………………………….. ………………………….. ……………… 6
1.2 Optical properties of nanostructures ………………………….. ………………………….. …………………. 8
1.3 Applications of nanostructures in developing of sensors ………………………….. ……………….. 10
Chapter 2 Ochratoxin A – Introduction ………………………….. ………………………….. …………………… 14
2.1 Specific Aptamer for Ochratoxin a A ………………………….. ………………………….. ………………. 16
Chapter 3 Fabrication of gold nanocups on PDMS – Experimental results ………………………….. ……. 19
3.1 Convective self -assembling fabrication method ………………………….. ………………………….. ……. 19
3.2 Gold nanocups substrate fabrication method ………………………….. ………………………….. ………… 21
3.3 Characterization ………………………….. ………………………….. ………………………….. …………………… 23
3.3.1 Atomic Force Microscopy (AFM) ………………………….. ………………………….. ………………… 23
3.3.2 Reflectivity ………………………….. ………………………….. ………………………….. ……………………. 24
3.3.3 Mercathophenil boronic acid detection test ………………………….. ………………………….. ……. 25
3.3.4 Amplification of SERS signal of thiophenol according to the nanocup size ………………… 26
Chapter 4 SERS Detection of Ochratoxin A – Experimental results ………………………….. ………… 29
4.1 SERS Detection of Ochratoxin A on rough Gold 5 nm ………………………….. ……………………… 29
4.1.1 Fabrication of rough gold 5nm ………………………….. ………………………….. …………………….. 29
4.1.2 Direct detection of OTA on rough Gold 5nm ………………………….. ………………………….. …. 30
4.1.3 Specific detection of OTA on rough Gold 5nm ………………………….. ………………………….. . 33

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4.2 SERS Detection of Ochratoxin A on flexible substrate of gold nanocups …………………………. 41
4.2.1 Direct detection of OTA on gold nanocups ………………………….. ………………………….. ……. 41
4.2.2 Specific detection of OTA on PDMS ………………………….. ………………………….. ……………. 42
4.2.3 Influention of the nanocup sizes in DNA detection ………………………….. …………………….. 43
Chapter 5 Conclusion ………………………….. ………………………….. ………………………….. ……………………. 44
Appendices ………………………….. ………………………….. ………………………….. ………………………….. ……… 45
Appendix 1. Raman spectroscopy ………………………….. ………………………….. ………………………….. .. 45
Appendix 2. Atomic Force Microscopy ………………………….. ………………………….. ……………………. 47
Appendix 3. Related to Thesis ………………………….. ………………………….. ………………………….. …….. 48
References ………………………….. ………………………….. ………………………….. ………………………….. ………. 49

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Thesis outline

Nanotechnology plays an increasingly important role in the development of biosensors. The
sensitivity and performance of biosensors are improved by using materials with nanoscale features
for their construction. The power of SERS lies in its ability to identify chemical species and obtain
structural information in a wide variety of fields including polymer and materials science,
biochemist ry and biosensing.
Ochratoxin (OTA) is a mycotoxin produced by various fungi, Penicillium verrucosum ,
Aspergillus ochraceus and Aspergillus carbonarius , which grow easily on food, especially cereals,
coffee beans, pork, and grapes. The need to develop hig h performing methods for OTA detection
with high sensitivity … able to improve the traditional ones is evident. A good approach in detection
of OTA is by using specific aptamers.
My thesis is focused on detection of OTA, development of SERS biosensor for th is type of
molecule and also fabrication of flexible SERS substrates.
Chapter 1, entitled Nanostructures – General Aspects – Literature , includes part 1:
Fabri cation methods of nanomaterials – which presents an overview of bottom -up and top -down
methodolo gies part 2: Optical properties of nanostructures – presents plasmonic fundamentals; and
part 3: Applications of nanostructures in developing of SERS based sensors .
Chapter 2, entitled Ochratoxin A – Introduction , includes the det ailed description of
Ochra toxin A and its toxicity . Second part contains the contains an introduction to aptamers.
Chapter 3, entitled Fabrication of gold nanocups on PDMS – Experimental results, includes
the fabrication method through convective self -assembling of the gold nanocup s substrate on PDMS
and the characterization of these substrates which were done in Cluj -Napoca .
Chapter 4, entitled SERS Detection of Ochratoxin A – Experimental results , includes SERS
Detection of Ochratoxin A on rough Gold 5 nm substrate and SERS Detect ion of Ochratoxin A on
flexible substrate of gold nanocups which was studied in Paris.
The final conclusions of my work are presented in Chapter 5.

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Chapter 1
Nanostructures – General Aspects – Literature

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Chapter 1 Nanos tructures – General Aspec ts – Literature

Nanoscale manufacture or nanofabrication consists in develop ing and controlling materials
with small dimensions from 1 to 100 nm . Many institutes of research have been working to improve
the fabrication methods for developing new nanometri c scale practical materials. Some of the most
used micro – and nanofabrication techniques include photolithography [1], soft lithography [2], film
deposition [3], etching [4], bonding [5], convective self-assembly [6], electrically induced
nanopatterning [7], colloidal monolayer [8] and focused ion beam lithography [9]. Nanofabrication
procedures offer the possibility for very reproducible fabrication of surfaces with complex
geometries and functionalities, which made them very used in the pharmaceutical and medical fields
as they, including novel medication delivery systems and biosensors. In addition, as a result of their
miniaturized size, these devices need less materials or reagents for analysis or operation, saving
money and time.

1.1 Fabrication methods o f nanostructures

Nanofabrication can be achieved in different ways depending on the approach. These
nanofabrication techniques are normally ordered into two principle classes: (1) bottom -up and (2)
top-down [1 0]. The difference between these two general c haracterizations is in light of the
procedures included in the manufacture of the nanometer -sized structures.
The top -down methodologies create nanoscale structures by controlled expulsion of
materials from bigger or mass solids. This technique includes sc aling down integrated -circuit (IC)
creation that has been standard since the 1970s to acquire particular nanoscopic elements,
lithography strategies (physical top -down) or chemical processes (chemical top -down) are used. The
most conventional physical top -down manufacture systems are based on the utilization of electrons
(Electron Beam Lithography, EBL), ions (Ion Beam Lithography, IBL) or photons (Optical
Lithography, OL). Additionally, a physical top -down creation strategy is NanoImprint Lithography

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(NIL) , a procedure which doesn't include any beam of particles, yet just a hard stamp that is utilized
to engraving nanoscale highlights onto a polymer film. Out of all the strategies introduced above,
OL is the most broadly utilized as a part of the manufactur e of nanoscale components, particularly
for integrated circuits.

The bottom -up manufacture idea utilized for the creation of nanostructures endeavors
straightforward and little building blocks like atoms, molecules or nanoparticles that are self –
assembled into bigger and more interesting structures. Most of the bottom -up manufacture systems
utilize chemically synthesized nanoparticles. Liquid phase methods are also numerous. It is within
the liquid phase that all of the science self -assembly and synthesis occurs. A standout amongst the
most widely recognized method utilized for the creation of solid substrates is the nano -sphere
lithography (NLS) [10], in light of the self -assembling of polystyrene or latex micro scale or nano –
spheres on solid layers follo wed by the deposition of a metallic thin film of wanted thickness and
consequent evacuation of the polystyrene spheres. Atomic layer deposition is a mechanical
procedure that is fit for covering any material, regardless of size, with a monolayer or to a gr eater
extent a slim film.
The principle advantages of the bottom -up technique is that desired nanoscale structures are
delivered in a less difficult, less expensive way and offer high throughput. Disadvantages include
establishment of long -range order. Bo th top -down and bottom -up manufacture methodologies may
be utilized to realize a range of all-around characterized nanostructured materials with alluring
physical and chemical attributes. Among these, the bottom -up self -assembly procedure offers the
most r ealistic solution toward the manufacture of next -generation useful materials and devices.

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1.2 Optical properties of nanostructures

In the most recent decade analysts done a serious work with a specific end goal to
comprehend the plasmonic fundamentals an d creating of plasmonic device based on nanostructures.
Researchers studied that surface plasmons can be found in diverse structures, for example,
propagative electron density waves at the interfaces between a dielectric and a metallic layer or
localized e lectron oscillation in metallic nanostructures or nanoparticles. Plasmonics represent a
relative new bearing that has as a ground state properties of collective electron excitation, which are
referred to likewise as surface plasmons, in films or nanostruct ure of noble metals (Au, Ag, Cu)
[11],[12]. Key examination of plasmonic uncovers that their interesting properties can be interpreted
in different useful applications, from identification at single molecule level to high resolution optical
imaging beneath diffraction point or upgrade transmission through a variety of subwavelength holes.
Plasmons are collective oscillations of the electron cloud of a metal, that is to say plasma
oscillations; The latter are quantified in the same way as photons or phonons, which leads to
defining them as quasi -particles. When these plasmons are excited at the plane interface between a
metal and a dielectric, this is called surface plasmon. Another interesting case is that of the metallic
nanoparticles for which the oscillat ion of the plasma takes place throughout the volume of the
nanoparticle rather than strictly at the metal / dielectric interface.
At a plane interface between a metal and a dielectric, such as a mirror, it is possible to excite
with photons a plasmon propa gating like an electromagnetic wave on the surface of the metal
(Figure 1 .1) And which is then called delocalized surface plasmon or propagating surface plasmon
(PSP).

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Hy
Figure 1.1: Representation of the propagation of a surface plasmon at the interface between a
metal and a dielectric of respective permittivity εm and εd, the field lines induced by the propagative
wave of the oscillation of the local charges are presented under Curved arrows. The projection of the
different components of the plasmon field is presented on the right, the electric field E is in the plane
(x, z) and the magnetic field H is along the y -axis.

In the event that the wavelength of the incident light is larger than the dimension of the
nanoparti cle, the electrical element of the radiation occurrence results in a dipolar distribution of the
electronic cloud. Because of its localized character, the electrical charge is going to oscillate at a
specific frequency. Despite what might be expected, if t he frequency of the incident light matches
the frequency of the surface electron oscillations, photons begin to interact resonantly with the
nanoparticle. As a consequence of this interaction, metallic element nanoparticles will show a strong
optical pheno menon called, absorption band, in the UV -Vis region, that isn't present w ithin the bulk
metal spectrum [13],[14],[15 ].
Surface plasmon resonance occurs when polarized light strikes an electrically conducting
surface at the interface between two media. The resonance condition is built up once the frequency
of incident photons matches the natural frequency of oscillatory surface electrons against the
restoring force of positive nuclei. LSPR excitation leads to wavelength -selective absorption with
extraordina rily giant molar extincti on coefficients ~10 -11 cm -1 [16 ] resonant Rayleigh scattering
[17],[18 ] with an associate degree to that of 106 fluorophores [19] and increased local
electromagnetic fields close to the surface of the nanoparticle [20 ].
Ez
Ex
Excitation
Lines of fi eld
Dielectric
Metal – + + – – + + – – + + – – + + – εd
εm

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1.3 Applic ations of nanostructures in developing of sensors

The excitation states of surface plasmons are in very sensitive changes within the dielectric
constant of surrounding medium. In these condition optical biosensors bases on plasmonic
transducers represent a powerful alternative in non -labeling analysis of biomolecular interaction
[21]. Notwithstanding the colossal aforementioned favorable circumstances, use of Kretschmann
pure mathematics for excitation of SPR makes an arrangement of inconveniences, for exa mple,
limitation of the SPR sensors to detection of a little array of biochemical response and therefore the
chance of creation in smaller than usual scale.
Keeping in mind the end goal to conquer these detriments, the enthusiasm of scientists in
optical nano -sensors in light of LSPR in metallic nanostructures has expanded [22],[23],[24],[25 ].
The instrument at the base of LSPR detecting is the alteration of the electromagnetic field decay
length after the absorption of molecules on the metallic surface. M ore particular on account of
metallic nanoparticles, the electromagnetic field has a working mode fundamentally the same with
the engendering of SPPs (Surface plasmons polariton) on a level metallic film and empowers the
transduction of chemical binding ev ents into a quantifiable wavelength shift of the extinction peak.
Regardless of exhibited proficiency of LSPR biosensors in light of nanoparticles in laboratory
[26],[27],[28 ], the manufa cture of biosensor devices based on nanoparticle and LSPR still remai ns a
troublesome errand and requires essential enhancements with respect to spatial resolution,
reliable ness and monodisperse in size and form of metallic nanoparticles.
Surface plasmons have been used for some time, to enhance the surface sensitivity of m any
spectroscopic analyses together with fluorescence and Raman scattering. The increase in force of the
Raman signal on specific surfaces happens as a result of an upgrade in the electric field gave by the
surface. At the point when the incident light in the experiment strikes the surface, localized surface
plasmons are excited. The field enhancement is greatest when the plasmon frequency is in resonance
with the radiation. For scattering to occur, the plasmon oscillations must be perpendicular to the
surface, in the event that they are in -plane with the surface, no scattering will occur. The subsequent
spectrum of Raman scattering comprises in a series of bands that are comparing to vibrational or
rotational transition particular to the structure of partic le, subsequently giving a certain chemical

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"unique mark" extremely valuable for analyze recognizable proof. In spite of the immense point of
interest of high specificity, the Raman scattering procedure is an extremely weak phenomenon as
simply approximatio n of 1 to 106 -1010 photons a re inelastically scattered [29],[30],[31 ].
Therefore, by utilizing plasmonic nanostructures, for example, gold and silver nanoparticles, with
plasmon resonances situated inside of the wavelength extent used to energize Raman mo des, that are
the visible and near -infrared regions of the spectrum, scientists had the capacity distinguish
chemical “fingerprint” of a solitary molecule because of increase the Raman signal up to 14 or 15
order of magnitude [32],[33 ],[34] .
Surface Enhanc ed Raman Scattering (SERS) is a phenomenon that was discovered in 1974
by M. Fleischmann et al. [35], who observed a very intense Raman signal of pyridine adsorbed on
the surface of a rough silver electrode. In the first place, the enhancement of the signa l was
attributed to the simple increase in specific surface area of silver under the laser beam, due to the
surface roughness of the electrode. However, such a hypothesis was not enough to explain such a
large increase in the signal, and it was only late r that the phenomenon was explained
electromagnetically [36,37 ] and chemically [38,39 ]. Thus, it can be seen that any molecule adsorbed
on a nanostructured metal surface has its Raman signal enhanced by a very important factor,
typically of the order of 106 and possibly up to 1012. Such amplification factors have made it
possible to detect and to observe single molecules. [ 40,41,42 ]
The power of SERS lies in its ability to identify chemical species and obtain structural
information in a wide vari ety of fiel ds including polymer and materials science, biochemistry and
biosensing, catalysis, and electrochemistry. The enhancement takes place at a metal surface which
has nanoscale roughness, and it is molecules adsorbed onto that surface which can undergo
enhance ment. Typical metals used are gold or silver – preparation of the surface can be through
electrochemical roughening, metallic coating of a nano -structured substrate, or deposition of
metallic nanoparticles (often in a colloidal form). Many researchers cr eate their own SERS
substrates, but commercially available kits offer a more routine approach. SERS is a highly sensitive
and sel ective technique for use in the detection of biological samples. SERS it’s a vast topic but in
terms of biosensing has been reviewed in great detail buy other researchers . SERS biosensors are
used in detection of various biological samples and diseases, including various cancers, glucose
detection and other. SERS biosensing has been used to detect and identify small molecules, nuc leic

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acids, lipids, peptides, and proteins, as well as for in vivo and cellular sensing. SERS detection of
biomolecules has been accomplished in both intrinsic and extrinsic formats. In intrinsic SERS
biosensing, the molecular signature for the analyte of interest, such as small molecule, DNA strand,
or protein, is acquired directly. In extrinsic SERS, the analyte or interaction of interest is associated
with a molecule with an intense and distinguishable Raman signature, traditionally a commercially
availa ble fluorescent dye, and it is the SERS spectrum of the tag that is used for sensing or
quantification. In either format, SERS has unique advantages for biosensing.

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Chapter 2
Ochratoxin A – Introduction

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Chapter 2 Ochratoxin A – Introduction

Ochratoxin A {N -[(3R) -(5-chloro -8-hydroxy -3-methyl -1-oxo7 -isochromanyl) carbonyl] -L-
phenylalanine} is a quite stable nephrotoxic molecule, able to resist to most food processing steps.
Aspergillus ochraceus and Aspergillus carbonarius are considered as the most frequent fungal
contaminants of several agricultural products. They are able to produce several polyketide -derived
secondary metabolites including mycotoxins such as Ochratoxin A (OTA), which has been found as
a common contaminant of cereals, coff ee beans, cocoa, nuts, beer, spices, dried fruits, animal organs
and also in certain wines, particularly those from Mediterranean area. The International Agency for
Research on Cancer , knowing the teratogenic and immunotoxic effects in humans [43, 44
Cruzaguado ], has classified OTA as a potential carcinogen (group 2B) for humans. At present,
regulatory limits for OTA exist in many countries, and testing of products is carried out at central
testing laboratories. European Union introduced limits for the leve ls of OTA in raw cereal grains (5
mg/kg), products derived from cereals (3 mg/kg), dried fruits (10 mg/kg), roasted coffee and coffee
products (5 mg/kg) and grape juice (2 mg/kg) (EC No 123/2005). The European Food Safety
Authority has also set a maximum l evel for OTA of 2 mg/kg for all types of wine.
Ochratoxin has been known for a long time as nephrotoxic [44,45,46] and the absorption of
contaminated food or prolonged exposure to a contaminated environment can lead to kidney failure
in children, man. This toxin is also genotoxic and is therefore potentially carcinogenic [47].
Moreover, its elimination is very slow, resulting in an accumulation effect in the case of repeated
exposures, which increases the associated risks.
Ochratoxin A Mw = 404 Da
Figure 2.1: Chemical formula for ochratoxin A

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The structure of ochratoxin A (OTA) is shown in Figure 2.1. We can see the presence of two
aromatic rings as well as a carboxylic group, an amide group and a ketone. All these groups will
offer an intense spectral sign ature because they have a strong Raman scattering cross section.
In most of the cases, to detect this specific molecule (OTA) involves conventional
techniques for analysis , such as high -performance liquid chromatography (HPLC), thin -layer
chromatography (T LC), and gas chromatography (GC), coupled to ultraviolet/visible, fluoresc ence
or mass spectrometry (MS) [48] . All of t hese procedure steps are very expensive and take a lot of or
too much time . Another problem is that protocols are still laborious and req uire trained personnel.
In the past decade s, immunochemical assays combined with new technologies have been
proposed for rapid quantitative or semi quantitative analysi s of OTA in food and beverages [49] .
They include enzyme immunoassays [50, 51], fluores cence polarization im munoassays [52] , and
immunosensors [ 53; 54; 55; 56]. In terms of stability and cost for analysis, the use of antibodies
remains a disadvantage even though it has good sensitivity and selectivity against OTA. The
creation of antibodies is linked to animal immunization and is a very laborious process that can take
several weeks. Another big problem is the dependence of temperature of antibodies , which in many
cases leads to degradation upon contact with surfaces, producing inconclusive data and results .
At present, ochratoxin is detectable using an ELISA [57] assay with a detection limit of 0.5
ppb and quantitation of the toxin concentration up to 10 ppb. This test is therefore perfectly suited to
quality control in the food industry. Such tests are available on the market and are commonly used.
However, ELISA tests take several hours to complete. Therefore, we propose to develop a SPRi /
SERS protocol that would be faster and that would allow in the near future simultaneous detection
of ot her toxins. In the ELISA test, the probe used to capture ochratoxin is a monoclonal antibody.
These antibodies are expensive and their stability is limited over time. In addition, these antibodies
have a very large size (15 nanometers) and cannot therefore be used in SERS because in this case
the target would be outside the zone of exaltation which is only a few nanometers. We therefore
decided to focus on a probe system called aptamer and consisting of an oligonucleotide. Objectives
for SERS specific Detec tion of Ochratoxin A (OTA) :
1) Substrate Au 5nm + 2) single strand DNA + 3) Ochratoxin A rough
surface

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2.1 Specific Aptamer for Ochratoxin a A

An aptamer is a single strand of DNA (ssDNA), also called an oligonucleotide. Aptamers are
developed in order to maximize their interaction with a target molecule. They can thus be
substituted for other probes and in particular antibodies or their fragments. The DNA
(deoxyribonucleic acid) consists of bases (Adenine, Cytos ine, Guan ine, Thymine (Figure 2.2 ),
linked by sugar, deoxyribose, and by phosphate groups. A single strand of DNA is therefore
characterized by a sequence of ATGC bases, shown in Figure 2.2.

Adenine Thymine Guanine Cytosine

Figure 2.2: Structures of the different bases composing the DNA

These bases are complementary and bind in pairs by forces of Van Der Walls. Adenine is
complementary to Thymine a nd Guanine of Cytosine. By virtue of these electrostatic forces, each
strand of DNA can bind to another complementary strand thus forming a double helix, discovered
by Watson and Crick [58]. There is a chirality in the structure of DNA, which makes it poss ible to
define a canonical sense of reading. A sequence begins at the free phosphate group located 5 'from
the deoxyribose which begins the sequence and ends with the free alcohol located on the 3' carbon
of the terminal deoxyribose. In order that the two helices can interlock and form a double helix, each
base is associated with its conjugate base and the order of the bases is reversed from 5 'to 3'. Thus,
on the example shown in Figure 2. 3, the complementary sequence of ACTG becomes CAGT. 7 6
5
8
9 4 1
2 3 4 5

6 3
2
1 7 6
5
8
9 4 1
2
3 4 5

6 3
2 1

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Phosphate

Desoxyribose

Figure 2.3 : Structure of a single strand of DNA of sequence ACTG (left) and pairing with its
complementary sequence CAGT giving a double strand of DNA (right).

For the detection of ochratoxin, an aptamer was selected by SPR method. Du ring these tests,
several sequences were used and the one with the greatest affinity with the target molecule was
chosen to be used within the sensor. This sequence is as follows and has been proposed in references
[59],[60]:
5’ GAT -CGG -GTG -TGG -GTG -GCG -TAA -AGG -GAG -CAT -CGG -ACA 3’ (36 nucleotides mw 11.5
kDa) Thiol en 5’ (C6).
Composition : A: 8/36 = 22% G: 17/36 = 47% T: 6/36 = 17% C: 5/36 = 14%. A chain of six
carbons, terminated by a thiol, is attached to the 5 'end of the aptamer. This end can th us cling to the
surface of the gold to functionalize our sample.

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Chapter 3 Fabrication of gold nanocups on PDMS
–
Experimental results

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Chapter 3 Fabrication of gold nanocups on PDMS – Experimental results

We create a new flexible substrate of gold nanocups on a PDMS base. In terms of fabrication
of the nanocups we used 2 steps. Firstly, we created the polystyrene monolayer through the well –
known method of fabrication convective self -assembly, after that we poured the PDMS liquid phase
and we let it dry. Next we pealed of the PDMS and the result was the nanohole imprint on which we
deposited the 50 nm layer of gold with physical vapor deposition method.

3.1 Convective self -assembling fabrication method
The initial phase in the manufacture pr ocedure is the self -assembling of micro or nano –
spheres in a monolayer on a solid substrate. Fischer and Zingsheim reported in 1981 surprisingly the
manufacture of a monolayer of 312 nm polystyrene spheres by deposing a colloidal solution on a
solid substr ate and letting it to evaporate. Amid the dissipation process just a little region of
monolayer was formed. Keeping in mind the end goal to create bigger range of monolayer of
polystyrene spheres the spin coating deposition method was used. The spin coatin g parameters, like
rotation speed and time, are vital and delicate in self -assembling particles in a controllable way.
Therefore, Nagayama et al. utilized a convective self – assembling method, namely the dip coating,
for creating a monolayer on a large are a [61],[62 ].
During the convective assembly, the dispersed particles are brought together and crystallized
in thin wetting films. By dragging the meniscus of a fluid colloidal suspension over a hydrophilic
surface at constant speed, the assembly is produce d. A compensation for the water evaporation at the
contact line area is created by the continuous flux of solvent from the thicker part of the layer.
Convective transport of particles toward the already ordered array causes the growth of an ordered
layer. This convective flow carries the suspended particles toward the 3 phase contact line. As the
layer of continuous 2D lattice grows, the substrate is withdrawn together with the already formed
layer. A schematic representation is shown in Figure 3.1:

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Figure 3.1: Schematic of the drying region of a thin evaporating film. Polystyrene monolayer grows
at the 3 phase contact line where water evaporates.

A homogeneous layer grows continuously if the withdrawal rate is equal to the rat e of layer
formation. At fixed evaporation rate and volume fraction, a uniform crystalline monolayer with a
defined thickness is obtained. Raising the substrate velocity will produce a monolayer or
diminishing the volume fraction of the suspension results in an incomplete layer. Conversely,
diminishing the substrate speed alternately expanding the volume fraction will result in the
formation of multiple layers. Besides substrate velocity and volume fraction, the growth velocity is
influenced by the evaporat ion rate which depends on the temperature of the colloidal suspension and
can therefore be controlled via the substrate temperature. Another important aspect in creation of the

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monolayer is the hydrophilicity of the glass surface. So before deposition we t reated the glass in
ultraviolet ozone in a cleaning system (PSDP -UVT, Novascan) for 20 minutes.
3.2 Gold nanocups substrate fabrication method

To obtain the polystyrene nanolayer we used 4 different colloids of PS with sizes: of 527 nm
(PS 527), 600 nm (P S 600), 719 nm (PS 719) and 836 nm (PS 836) diameters. The depositions were
obtained at room temperature as we did not have any cavity to close over the deposition machine.
We only controlled the speed of the deposition with the velocity of 20 -30µm/s of th e bottom glass
and the temperature of the bottom layer (20 -25˚C). We fluctuated the speed to obtain the best largest
surface of monolayer polystyrene.
The next step was to pour over slowly the PDMS elastomer mix in liquid form over the glass
with polystyrene nanolayer filling all the voids in the monolayer. We used a ratio of 10:1 of polymer
precursor and curing agent and we cured in an oven at 60˚C for 1 hour. After that we cut the edges
of PDMS and peeled it of gently so that only the PDMS comes off and the polystyrene remains
attached to the glass surface. The result was a nanocups array surface engraved in PDMS. You can
see the steps of production in Figure 3 .2:

Figure 3 .2: Schematic illustration of fabrication steps of the flexible 3D gold nanocups plat form,
Steps: (A) Deposition of PS on glass substrate, (B) Pouring PDMS onto the deposited PS, (C)
Peeling -off the PDMS imprint, (D) Obtaining the flexible PDMS substrate with impregnated
nanocups, (E) Coating the as -prepared flexible nanocups with gold fil m to obtain flexible plasmonic
substrate.

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Third and final step was the gold coating of the top side of the PDMS mold with a thick layer
of 50nm gold. The procedure was by thermal evaporation in high vacuum using a Prevac deposition
equipment (Prevac, Pola nd).

As a result, we obtained a large -area flexible ordered gold nanocups array platform. In
Picture 3 .3 you can see how the nanocups reflects and transmits light under the optical photo
camera. You already can see the dispersion of light on thin films.

Picture 3 .3: Left photo: nanocups on PDMS see through, Right photo: bending of PDMS and
beautiful light dispersion.

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3.3 Characterization
3.3.1 Atomic Force Microscopy (AFM)

The next step was to see the results of our work. We chose first the AFM technique where with help
of my colleagues from ICEI we obtained images of two different samples 600nm and 719nm. The
images were obtained with an Atomic Force Microscope system (Alpha 300 A, Witec). We can
observe that the grating remained the same of the polystyrene nanolayer in Picture 3 .4. The distance
between 2 centers of holes is the polystyrene size, so 600nm and 719nmfrom center to center. We
also can see that we have some imperfect lines that are higher which is due to the PS deposition,
which we k now that it deposes in sectors which has some zones where the deposition has gaps

AFM 719nm AFM 3D 719 nm AFM 600nm

Picture 3 .4: AFM images of the two sizes of nanocups (719 and 600nm)

In terms of gold deposition, we conclu ded that the gold deposited on the walls of the holes
and it thickened them, but also deposited in the bottom of the hole. A SEM image of a slice should
have been done so that we can see also the real depth of the wholes and the height of the walls of the
nanocups.

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3.3.2 Reflectivity

The experimentally measured optical reflection spectra of the obtained PS 527, PS 600 and
PS 719 nan oplatforms are shown in Figure 3 .5. Specifically, Figure 3.5 shows that the optical
responses of these 3D gold nanocups ar rays can be spectrally tuned by changing the diameter of the
nanocups, an important feature that should be taken into consideration for an optimal SERS
detection of analytes of interest. While a flat Au film exhibits the well -known transparency in green
and high reflectivity at longer wavelengths (see Figure 3 .5), in the case of our fabricated substrates,
localized and propagating SPR modes are intercoupled, being difficult to estimate how much
contribution comes from each mode. To note that free PDMS subst rate (non -coated with gold film)
is transparent and consequently no absorption band was recorded. Spectrums were acquired with
Spectrophotometer UV -VIS (V -530) with reflectivity Module (SLM -468S).
400 600 80005101520253035
Au 527 nm
Au 719 nmgold filmAu 600 nmReflectivity
wavelenght nm

Figure 3 .5: Reflectivity spectr a of gold film and nanocup size of 527nm, 600nm and 719

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3.3.3 Mercathophenil boronic acid detection test

At the beginning of my experiments, after I obtained the gold nanocups, me and my
professor wanted to do a detection test to see if the surface is go od for detection. We decided to use
a solution of 1mM MBA (4 -mercathophenil boronic acid) as is a good molecule that is hard to detect
through Raman, but can be easily detected with SERS and has already been studied in our
laboratory. As we can see in Figu re 3.6 after the addition of the MBA the higher peaks of PDMS
disappeared and we could see the most important bands of MBA at 1074 cm-1 and 1573 cm-1 as it is
shown in literature. We used the sample with nanocups 836 nm.
200 400 600 800 1000 1200 1400 16000500100015002000250030003500
SERS MBARaman PDMS Intensity (a.u.)
Raman shift (cm-1)

Figure 3.6: Raman spectra PDMS(red) and MBA Mercathophenil boronic acid(black) on nanocups
type 836nm

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The band at 1573 is visible through Raman also, but the band of 1074 cm-1 can be seen only
through SERS. So we concluded that we have surface amplification an d that the substrates are what
we wanted to create. The only problem that we did not consider is that this molecule is one that
gives good distinctive peaks and they have high intensities. But for molecules as OTA and DNA
where there appear a significant n umber of additional peaks with lesser intensity we had a much
difficult way to identify the specific peaks of our molecules. Spectrums were acquired with a Raman
mobile spectrometer Micro Raman System (R -3000, Ocean optics) with a 785 nm laser.

3.3.4 Ampl ification of SERS signal of thiophenol according to the nanocup size

On my first week in Paris I tested the detection of thiophenol on my 4 sizes of nanocups
substrates to see if it can detect it and to see maybe which is better in amplification. As a res ult, we
can see in Figure 3 .7 the spectra is almost the same. We get all the thiophenol major bands in all 4
spectrums which are an average of 10 acquisitions.
Raman shift (cm-¹)Intensity (counts)
500 1 000 1 50005001 0001 5002 0002 5003 0003 500 836
719
600
527

Figure 3.7 : SERS spectra of thiophenol on the 4 nanocup surface sizes (527,600,719,836)
baseline subtracted.

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The SERS spectra were acquired on a spectrometers LabRam 300 at 633 nm. The signals
were collected using a 80× objective. Acquisition time for one spectrum was 60 s with 200 μW
power. For each diameter, measurements were done ten times on different points to analyze the
reproducibility of the signal. SERS signals were then averaged and in terms of signal amplification,
we can see that the best amplification we get with the 527nm size surface and the 719nm one. The
SERS signal strength in dependence of the s ize of nanocups can be seen in Figure 3 .8:

Figure 3.8 : SERS signal according to the size of nanocups

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Chapter 4
SERS Detection of Ochratoxin A
–
Experimental results

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Chapter 4 SERS Detection of Ochratoxin A – Experimental results

4.1 SERS Detection of Ochratoxin A on rough Gold 5 nm
4.1.1 Fabrication of rough gold 5nm

Firstly, we made a SERS substrate, which does not require any lithography technique and
consists of a standard glass microscope slide on which we deposited 5 nm gold b y thermal
evaporation. As the gold badly wets the glass, nano -islets of gold are formed on the surface of the
lamella (Figure 4.1) forming a SERS substrate. The reason for using such substrates is that they are
easy to produce, that their manufacturing cos t is low and that they are very simple to use. Thus,
these substrates are adapted to the numerous tests necessary for the definition of the
functionalization protocols. We have therefore turned to this choice in order not to have to use many
lithographed s amples w hich are longer to manufacture.

Figure 4.1: SEM image of a rough gold film used as SERS substrate.

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4.1.2 Direct detection of OTA on rough Gold 5nm

Before carrying out a detection test, we wanted to characterize the t arget molecule in Raman
and SERS. The first test was taken by Raymond Gillibert a colleague of mine from University Paris
13. We have therefore performed a Raman measurement of the solid ochratoxin powder and we
have also deposited a drop of 1 mM ochratoxi n solution on a rough gold substrate before rinsing the
latter and performing a SERS measurement. The spectra are shown in Figure 4.2, 4.3. The SERS
spectrum and the Raman spectrum have common bands and the bands are widened in SERS.
200 400 600 800 1000 1200 1400 1600 18005k10k15k20k
Raman Intensity cnt
Raman Shift (cm-1)

Figure 4 .2: Raman on powder .

The SERS and Raman spectra were acquired on a spectrometer from Horiba Scientific: a
Xplora at 660nm. The SERS signals were collected using a 100× objective with a numerical
aperture of 0.9. Acquisition time for one spec trum was 60 s with 200 μW power. Measurements
were done ten times on different points to analyze the reproducibility of the signal. SERS signals
were then averaged (Figure 4.3) .

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200 400 600 800 1000 1200 1400 1600 1800-1000100200300400500600700800
SERS intensity (a.u.)
Raman Shift (cm-1)
Figure 4 .3: SERS on rough gold

We deposited an och ratoxin solution of 10 μM concentration on the rough gold surface
functionalized with the aptamer. The ochratoxin was diluted in 1M KCl solution, so that the solution
imposed high ion pressure on the aptamer, leading to its unfolding. An incubation time of one hour
has been applied so that the ochratoxin molecules have time to cling to the aptamer. The sample was
then thoroughly rinsed with distilled water to remove any non -specific adsorption of ochratoxin
molecules on the substrate, as well as saline buff er. The sample was then dried and characterized in
SERS.
After that we tried to see if changing the dilution of ochratoxin A can deliver changes to the
spectra in any way. In addition to KCl, we used other buffers to solve the ochratoxin, we tried H 2O
and TBS. In terms of concentrations used same the same parameter of 10 μM . The spectrums can be
seen in figure 4 .4:

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200 400 600 800 1000 1200 1400 1600 18000500100015002000250030003500 SERS intensity (a.u.)
Raman shift (cm-1)Raman OTA Powder SERS OTA SERS OTA+H2OSERS OTA+TBS
Figure 4 .4: Spectra of OTA directly on Au 5nm surface. Black: Raman on powder, Red: SERS from
literature, Green: OT A in H2O buffer, Blue: OTA in TBS buffer.

The SERS OTA an d Raman OTA Powder are spectrums obtained by my colleague and
coordinator Raymond Gillibert in Paris . He was already studying the detection when I arrived in
Paris in 1 November 2017. I will be usin g them few more other times as a comparison factor with
my results and his. We can see that the SERS spectrums look almost the same. SERS of Raymond
was also done in water buffer .
The result shows us that the molecule can be detected on the gold 5nm rough surface. We
can see the bands of ochratoxin A from 1562 cm-1 and 1268 cm-1. We can see also the bands from
566 cm-1 and 960 cm-1 but are much lower.

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4.1.3 Specific detection of OTA on rough Gold 5nm

Step 1. Detection DNA different buffers
In a first s tep, the surface of the rough gold film was functionalized with the aptamer, by
dropping a drop of concentrated solution at 10 μM overnight before rinsing with distilled water. A
schematic diagram of DNA on surface is presented in Figure 4 .5.

Figure 4 .5: Schematic diagram of the surface functionalization of our SERS substrate. The
aptamers will allow the capture of the targ et molecule on the surface.

The spectra plotted in Fig ure 4 .6 are derived from quantum molecular simulations using the
Density Functional Theory (DFT). They were carried out by Shivalika Tanwar, PhD student in the
CSPBAT laboratory, working on molecular m odeling and crystallography. We used these spectra as
a basis for interpretation of the Raman bands of aptamers. Another bibliographic source corresponds
to the work of C. Otto et al. [63] (1986) which propose a detailed assignment of the different bands
of the DNA bases visible in Raman and SERS. Aptamer
S S S S S S S S S
SS
Gold surface

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Figure 4 .6: Raman spectra simulated by DFT of the bases adenine, cytosine, guanine,
thymine.

Finally, the work of B. Prescott et al. [64] allowed an analysis of the spectra of strands of
DNA in different conformations. We used these results to perform an allocation of the most intense
bands of our spectra ( Table 4 .1). The broad band, visible at 1665 cm -1 corresponds to the elongation
vibrations of the ketones of bases T, G and C.
ν (cm-1) Bases allocated Vibration
1573 G N3-C4-C5
1483 G N1-C2 et N 1-C6
1327 A / G N7 entre les C 5 et C 8
1093 Phosphate ν(PO 2-)
1011 Desox. -Phosp. P-O-C, antisymmetric stretching
782 C / T Ring breathing mode
735 A Ring stretching
667 T/ G / A
Table 4 .1: Attribution of the main Raman bands of our aptamers
200 400 600 800 1000 1200 1400 1600 1800 Cytosine
5000

Adenine

0 10000 15000 20000
Guanine Intensity (a.u.) Thymine
Raman Shift (cm-
1)

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If the SERS and Raman spectra of the ochratoxin aptamer are compared, a large number of
spectral differences can be observed. Indeed, we see in SERS the disappearance of certain bands. To
explain such differences, it can first be assumed that the DNA strand undergoes a conformational
change when it is dried and covalently attached to the gold surface. It can thus be understood that
there is a slight shift of the Raman bands as well as a chang e in their relative intensity. But this
hypothesis cannot explain so great a difference between spectra. We know that our DNA strand
making 36 nucleotides has an unfolded length of 12 nm (about one third nanometer per base [65]).
However, the SERS intensit y decreases exponentially when the molecule moves away from the
surface over a distance of the order of a few nanometers. Thus, if the DNA strand is orthogonal to
the surface, only the first bases of the strand will be visible, which explains the reduced n umber of
Raman bands observable on the SERS spectrum. In more detail, it can be seen that the 1483 cm -1
band attributed to the guanine disappears completely. This behavior can also be explained by the
fact that the selection rules in SERS are different fro m those in conventional Raman scattering. The
disappearance of this same band was observed by C. Otto et al. [63] on guanine alone. Guanine
being the first base of our chain and composing 47% of our total sequence, this result was expected.
The disappearan ce of the phosphate bands ν (PO2 -) at 1093 cm -1 is also observed. Since it is a
charged group, it is not surprising that the chemical interaction with gold drastically changes the
behavior of the band. The band at 782 cm -1, for its part, shifts towards th e higher frequencies and
decreases in intensity.

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200 400 600 800 1000 1200 1400 1600 1800050010001500200025003000
SERS DNA refDNA+KCl
DNA+TBS
DNA+HEPES
DNA+PBS
DNA+H2OSERS intensity (a.u.)
Raman shift (cm-1)
Figure 4 .7 First set of buffers for the DNA

In figures 4 .7, 4.8 We did two sets of tests of different buffers for the aptamer connection to
the surface. In the first set we used H2O, PBS (phosphate -buffered saline), HEPES (4 -(2-
hydroxyethyl) -1-piperazineethanesulfonic acid), TBS (tris -buffered saline) and KCl ( potassium
chloride ) and in the second test H2O, PBS (phosphate -buffered saline) and DSB. We used 20µl
drops to cover the g old rough surface. We let it sit for 1 hour and after that we rinsed with water and
dried the surface with NO 2. We did this to see which helps better the aptamer to bind to the gold
better. But we came to the conclusion that just using water is ok, because the buffers included some
major modifications in the spectra of the aptamer.

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200 400 600 800 1000 1200 1400 1600 180007001400210028003500420049005600
SERS DNA refSERS intensity (a.u.)
Raman shift (cm-1)DNA+DSB
DNA+PBS
DNA+H2O
Figure 4 .8 SERS spectrums of the s econd set of buffers for the DNA

Step 2. Specific detection OTA

The next step in the detection of Ochratoxin A using rough gold surface was the
addition of the ochratoxin over the sets of aptamer funcionalised samples. We used 20µl drops to
cover all the surface of the rough surface. We let it sit for 1 hour and after that we rinsed with water
and dried the surface with NO 2. On this step we tried OTA in HEPES and H2O buffers. Figure 4 .9,
4.10.

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200 400 600 800 1000 1200 1400 1600 1800050010001500200025003000350040004500SERS intensity (a.u.)
Raman shift (cm-1)KCl DNA+ OTA HEPES
TBS DNA+ OTA HEPES
HEPES+DNA + OTA HEPES
PBS+DNA+ OTA HEPES
H2O DNA+ OTA HEPES
SERS DNA+OTA ref
Figure 4 .9 SERS spectra of Ochratoxin in HEPES added over the DNA

We see that in the second step Figure 4.10 , we have better signal. We were think ing that
maybe the cause of this is because the DNA and OTA from the first step was removed from frige –
20˚C for more than a month. We know that DNA degradates in time if it is not kept in the fridge. So
the second set aquisition came from this idea that w e should try to use a new fresh set of aptamer
and OTA.

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200 400 600 800 1000 1200 1400 1600 18000100020003000400050006000SERS intensity (a.u.)
Raman shift (cm-1)PBS+DNA + H2O+OTA
H2O+DNA + H2O+OTADSB+DNA + H2O+OTA
SERS DNA+OTA ref
Figure 4 .10 SERS spectra of Ochratoxin in H2O added over the DNA

The SERS spectra were acquired on the spectrometer from Horiba Scientific, an Xplora at
660. The signals were collected using a 80× objective . Acquisition time for one spectrum was 60s
with 200 μW power. For each diameter, measurements were done nine times on different points to
analyze the reproducibility of the signal. SERS signals were then averaged
In Figure 4 .11 we have the comparison of the spectrums obtained by me in H2O solvent in
comparison with the ones already obtained by my colleague. We have the spectra of Ochratoxin
direct on sur face, and the two subsequent of DNA on substrate and DNA plus OTA added. This way
we can see which bands are from the DNA and which are fro m ochratoxin in the final spectra.

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200 400 600 800 1000 1200 1400 1600 18000100020003000
OTA-H2OSERS intensity (a.u.)
Raman shift (cm-1)SERS DNA+OTA refOTA-H2ODNA-H2ODNA-H2O+OTA-H2O
Figure 4 .11 SERS spectrums of OTA and DNA alone in comparison with SERS of
ochratoxin over DNA final spectra in H2O solvent .

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4.2 SERS Detection of Ochratoxin A on flexible substrate of gold nanocups

4.2.1 Direct detection of OTA on gold nanocups

Before carrying out a detection test, we wanted to characterize the target molecule in Raman
and SERS with the gold nano cups on PDMS . We have therefore performed a SERS measurement , a
drop of 1 m M ochratoxin A solution was deposited on a gold PDMS substrate. An incubation time
of one hour has been applied so that the ochratoxin molecules have time to cling to the gold surface.
The sample was then thoroughly rinsed with distilled water to remove an y non -specific adsorption
of ochratoxin molecules on the substrate. This spectra + SERS of rough 5nm gold are shown in
Figure 4.12. The result shows us that the molecule can be directly detected on the gold nanocups.
We can see the bands of ochratoxin A fr om 1562 cm-1 and 1268 cm-1 with high intensity.
200 400 600 800 1000 1200 1400 1600 180002004006008001000SERS intensity (a.u.)
Raman shift (cm-1)1mM-OTA on nanocups
SERS OTA ref

Figure 4.12 : Spectra of 1mM -OTA on nanocups, compared with reference spectra of OTA on gold
5nm

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The SERS spectra were acquired on the spectrometers from Horiba Scientific: Xplora a t 660.
The signals were collected using an 80× objective. Acquisition time for one spectrum was 60 s with
200 μW power. For each diameter, measurements were done 10 times on different points to analyze
the reproducibility of the signal. SERS signals were t hen averaged.

4.2.2 Specific detection of OT A on PDMS
After we saw that the detection with gold nanoholes can be done, we turned to the second
part of the experiment. We first treated the substrate in UV ozone, after that we functionalized the
surface fir st with a solution of 10µM of sing le strand DNA in water. After a time -lapse of 1 hour we
rinsed with ultr apure water and did the spectrums on Horiba microscop e. Then we added the
ochratoxin for another hour. In Figure 4.13 we have the spectra of the sampl e before and a fter the
addition of ochratoxin . I also added the spectra of OTA directly on nanocups surface and the spectra
obtained on Au 5nm rough surface. This way we can easily c ompare the peaks of ochratoxin.
400 600 800 1000 1200 1400 1600 180006001200180024003000
DNA-H2O+OTA-H2O
DNA-H2O
OTA-H2OSERS intensity (a.u.)
Raman shift (cm-1)SERS DNA+OTA ref

Figure 4.13 : Comparison between: Red: SERS spectra of OTA direct on surface – Green: SERS of
DNA on substrate – Blue: SERS OTA after the DNA on nanocups – Black: SERS of OTA on Au
5nm as reference

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The result shows us that the molecule can be detected with the nanocoups . We can see the
bands of OTA from 1562 cm-1 and 1268 cm-1. We can see also the bands from 566 cm-1 and 960 cm-
1 but are much lower.
4.2.3 Influention of the nanocup sizes in DNA detection

As we wanted to precisely see if the sizes of PS spheres influe nce the detection of
DNA and to be able to better detect the DNA we did an additional experiment with all
the 4 sizes. We could determine that the bands of DNA could be seen in all 4 samples,
but we decided to continue in experiments first with 719 size be cause I had more samples
and also with 836nm. Compared to the first approach with the Au 5nm we had better
intensities and the specific peaks of DNA 1093cm-1, 1327cm-1 and 1573cm-1 could be
easily identified in Figure 4.14 .
400 600 800 1000 1200 1400 1600 1800050100150200250300350400450500550600650700750800850
SERS DNA refSERS intensity (a.u.)
Raman shift (cm-1)836-PBS+DNA
719-PBS+DNA
600-PBS+DNA
527-PBS+DNA

Figure 4.14 : SERS spectra of DNA on all the 4 ty pes of nanocups in comparison to reference.

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Chapter 5 Conclusion

In this dissertation we have seen that ochratoxin is a highly dangerous mycotoxin whose
concentration must be regulated and controlled in any pr oduct that can contain it. Despite the
existence of an ELISA test, we have chosen this target because it represents a major interest for
quality control in agri culture -food and we wish to validate the instrument developed for a project in
future for the de tection of toxins. We propose a detection with an aptamer, due to its high level of
sensitivity and specificity allowing a better response in SERS. We have characterized the chosen
aptamer in classical Raman and SERS. The aptamer selected in this study can potentially be used as
a recognition element for the development of OTA specific biosensor.
Finally, we have demonstrated that it is possible to detect ochratoxin by SERS method over
two types of substrates. This detection has been demonstrated on gold ro ugh film substrates on glass
for reasons of simplicity and availability of substrates. The other method implied a more laborious
method but the gold nanocups were formed on a flexible PDMS substrate that can have many
biological ap plications . We can also s ee that we do not have a real identification of the target
molecule but that its detection is done by observation of modification of the spectrum of the
aptamer. We also could detect other molecules like Mercathophenil boronic acid which can be
used in oth er biosensing applications .

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Appendices

Appendix 1. Raman spectroscopy

Raman spectroscopy is a useful technique for various chemical analyses. In Calcutta on
February 28, 1928, Professor C. V. Raman was the first to observe the scattering of light at di fferent
wavelengths not present in the incident light source when a monochromatic incident light beam was
applied on a sample. The Raman spectroscopy is a spectroscopy technique used to observe
vibrational, rotational and other low – frequency modes of the system. Electric and magnetic
components of a radiation cause the electrons to be in the intense field, at the point when
electromagnetic radiation touches the molecule, and interacts with the electron cloud and the bonds
of the molecule. In this way the electrons of molecule/particle will oscillate with the same frequency
as the incident radiation.
The emanated radiation is elastic scattered radiation and is known as Rayleigh scattering.
When electrons of a molecule are excited by a low -energy incident be am to a virtual excited state
below the next -highest electronic state and fall back to another vibrational sate different from the
incident one, takes the Raman scattering place. Common are the transitions from a vibrational state
to another. Thus, the pho tons emitted by relaxation of the electrons have completely different
energies and therefore different wavelengths compared to those of incident excitation beam.
In Raman spectroscopy, the sample is irradiated with an intense monochromatic beam
(laser). Th e incident wavelength does not have to be one that is absorbed by the molecule, although
it can be, as in the case of resonant Raman scattering. Excitation is considered to take place to a
virtual level, which is not the actual level of the molecule (Figur e A1.1) and corresponds to the laser
frequency.

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Figure A1.1: Energy -level diagram showing the states involved in Raman signal. The line thickness
is roughly proportional to the signal strength from the different transitions Reproduced from [66 ].

The bas ic components of Raman system include a light source (laser), collection optics to
gather the Raman – scattered light, and detection system (Spectrometer, CCD). The advantages of
confocal microscopes are: increased resolution, by using spatial pinhole to e liminate out – of – focus
light in specimen that is thicker than the focal plane, thus the light rays from outside the focal plane
will not be recorded; possibility of recording three dimensional data sets; focus on sample. The
combined Raman spectrometer with confocal microscope presents the advantages of taking
complete Raman spectrum at every image pixel, extraction of relevant information (peak intensity,
position etc.), and the display of taken information as image. Figure A1.2 presents the main
compon ents of the confocal Raman system used in our lab, from Witec, Alpha 300 R model, and the
picture of the system.

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Figure A1.2: The main components of the confocal Raman microscope 300 R and the beam path
(right) and picture of the confocal micro scope (left) Reproduced from [67],[68 ].

Appendix 2. Atomic Force Microscopy

The Atomic Force Microscopy (AFM) was invented in 1986 by Binning, Quate and Gerber.
Since then the AFM has evolved into an invaluable surface analysis technique on micro – and
nanoscales and even on atomic and molecular scales. With the help of AFM it is possible to image
the molecular structures of real -space surfaces and even additional surface properties such as
adhesion, stiffness, magnetic properties, conductivity and many more.
The o perating principle of the AFM is based on scanning the surface of a sample using a
sharp tip. In order to trace the surface topography are used various interactions between tip and
sample as feedback mechanisms. AFM’s most widespread operating modes are th e contact mode
(CM) and the tapping mode (AC). In CM, a sharp tip (less than 10 nm across) is brought into contact
with the sample while the repulsive force between tip and sample bends the cantilever. With the help
of a highly focused beam deflection syst em the bending of the cantilever can be measured. (see
Figure A2.1)

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Figure A2.1: Principle of AFM c ontact mode. Reproduced from [69 ].

By keeping the bending of the cantilever constant, a constant force is applied to the sample
while scanning the tip acr oss the surface. The up and down movement of the scan stage is recorded
as surface topography. These images highlight the edges of various topographic levels. Beside these,
invaluable information is also provided by recording the torsion of the cantilever. The disadvantage
of contact mode is in samples which are too soft or weakly bound to the substrate. In AC Mode this
can be overcome where the cantilever is oscillated at its resonant frequency with free amplitude. By
keeping the damping of the amplitude c onstant, the surface topography can be imaged.
Appendix 3. Related to Thesis
A. Sabau, C. Leordean, Monica Potara, Sanda Boca -Farcau, C. Farcau, S. Astilean,
Fabrication of parallel plasmonic stripes through convective self -assembling of gold nanoparticles –
Advanced Spectroscopy on Biological and Nanostructured Systems, 7 -10th of September 2014,
Cluj-Napoca (Romania)

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for detection of Ochratoxin A
49
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