Toward an Optofluidic gene Sensor based on Fluorescence Enhancement in a Hollow Bottle Microresonator

6 
 
Introduction 
My thesis work was carried out at the Optoelectronic Research Centre (ORC) in the 
United Kingdom. Founded in 1989, the ORC is situated within the Faculty of Physics 
and Applied Science of the University of Southampton and is considered, in the interna-
tional scientific community, as one of the leading institution in the field of photonics 
and optoelectronics. Its facilities include the EPSRC (Engineering and Physical Scienc-
es Research Council) Centre for Innovative Manufacturing in photonics, which offers 
the opportunity to the institute to develop fibres and materials that can meet market 
needs, and to identify commercially viable ways of manufacturing them. New genera-
tion of fibres are the main key of research, used for every application such as communi-
cations, laser manufacturing, sensing, infrared fibres and defence countermeasures. For 
this reason, it supports the growth of companies, not only in the UK (as for SPI Laser 
company of Southampton), but all over Europe and even in Italy as for Selex Galileo 
and Pirelli, at least before the telecommunication bubble. 
The project was started in the Optical Biosensors and Biophotonics Group that is more 
oriented to the area of micro and nanofabricated devices, for bio-analyses or for bio-
medical applications, either based on optics, microfluidics or self-assembled techniques. 
The project, due to its multidisciplinary characteristic, was continued within the Inte-
grated Photonic Devices Group that traditionally exploited mainly planar photonic de-
vices, for various applications included diagnostics in medicine, but, more recently, also 
integrated microsphere resonators fabricated with fibre technologies. The project meets 
the demand of small integrated biological sensors which could eliminate the lengthy and 
hard laboratory work traditionally required to perform gene detection. For these devices, 
the biochemical functionalization of the device is the first aspect to consider, which con-
fer to the optical device its specificity, i.e., the way to interact with biological entities. 
For optical methods the sensitivity and speed is considerably enhanced respect to elec-
trochemical approaches; moreover, optical detection can be integrated with a capillary 
fabricated with fibre technology that provides the advantages of microfluidics. For bio-
logical measurements, optics offers different approaches. In particular, label-free meth-
ods are very interesting since do not require the use of any marker and achieve among 
the best sensitivity in biosensing. Unfortunately, the label-free technique was found not 
suitable for the fluidic device considered and it was necessary to consider the use of flu-
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orescent markers. A characterization of the device was carried out in order to verify the 
principle of optical transduction: it was aimed to the spatial and spectral visualization of 
the resonances by the excitation of specific fluorophores. 
In first chapter the widely used method for detection of nucleic acids strings, known as 
hybridization, is presented with the explanation of the basics of the nucleic acids biolo-
gy. 
The optical approach exploited in this work requires an understanding of the concept of 
fluorescence that is the physical effect that provides the signal and is explained in chap-
ter 2. 
Chapter 3 is dedicated to the state of the art in biosensing, illustrating basics, require-
ments and several possible approaches, focussing on optofluidic biosensors. 
The state of the art specifically relevant for the optical resonator device used in this 
work is described in chapter 4 after summarizing its physics principle. 
Materials exploited in this work came from many disciplines and are listed in chapter 5, 
in addition to the chemical methods employed for the research and the optical scheme 
used for measurements. 
The experimental work is reported in chapter 6 where the functionalization of the de-
vice with the bioreceptor and the characterization of the performance of the transducer 
are described in detail.
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1 Nucleic Acid Hybridization Assay 
Nucleic Acid Hybridization Assay is a successful assay method in molecular genetics as 
it allows the identification of specific genes from the whole genome of a species. There-
fore, it is widely used for bacteria and virus detection, forensic tests, environmental 
monitoring, food quality control and biomedical diagnoses [1]. This chapter explains 
principles of hybridization assays and its methods for application, but, first of all, it is 
necessary to introduce the basis of molecular biology focusing on the behavior its enti-
ties. 
1.1. Nucleic acids features 
Nucleic acids are two groups of biomolecules which encode information about the func-
tion of cells in which they are located. Their structure, shown in figure 1.1, is a polymer 
of nucleotides that consists in a chain of sugar and phosphate molecules, each associated 
to one of the nucleobases: Guanine (G), Cytosine (C), Adenine (A), Thymine (T) and 
Uracil (U). 
 
Fig. 1.1 - Nucleic acids structure [2]. 
These nitrogen-based molecules have the role of symbols for encoding the genetic in-
formation. It is known that very electronegative atoms such as fluorine, oxygen or ni-
trogen provide strong hydrogen bonds; as a consequence the chemical composition of 
nucleobases causes them to create pairs. This bond becomes very specific depending on 
the number and position of electronegative tails; therefore cytosine links exclusively 
with guanine while thymine and uracil are both suited for adenine because they are very 
similar in the structure.
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The acid which stores information inside the nucleus is named Deoxyribonucleic Acid 
(DNA) and is composed of sections named genes. These are formed by two long com-
plementary oligonucleotides strands with their typical double helix shape. The second 
group of nucleic acids is named Ribonucleic Acid (RNA) and it is used to transfer in-
formation between cell organelles with the aim of generating proteins. Concerning the 
structure of the two acids, their backbone is composed by different sugar molecules; in 
addition, thymine provides the conjugation with adenine in DNA, while it is totally re-
placed by uracil in RNA. Because of its temporary transport function, RNA shows only 
a single strand helix shape inside the cell, but it also results in the creation of coupled 
strings [3]. 
1.2. Nucleic acid thermodynamics 
The non-covalent bond between nucleobases allows any combination of complementary 
nucleic acid strings to join together, whether they are of the same type or not. This pro-
cess, known as annealing or hybridization, produces double-strings called duplexes or 
hybrids. Nucleic acid thermodynamics also includes a process for splitting duplexes, 
known as denaturation. The process is usually optically monitored as the absorbance, at 
the wavelength λ = 260nm, in free nucleotides is higher than in duplexes; alternatively, 
denaturation is identified by measuring the heat capacity by differential scanning calo-
rimetry. The most common and oldest method used in the laboratory consists in increas-
ing the duplexes temperature above the so called melting point and rapidly cooling the 
sample to trap the strands in the separate state. A useful tool for studying denaturation is 
the melting curve, of which figure 1.2 is an example, which shows separation of du-
plexes as a function of temperature. In this way the threshold is clearly defined as the 
temperature required achieving denaturation of half the duplexes; for a complete separa-
tion of all the duplexes, a 20-30°C increase is usually necessary. The thermal energy re-
quired to separate two strands is dependent on many factors. First of all, the length of 
the duplex means more hydrogen bonds to split. The second factor is the base composi-
tion: while adenine linking makes just two hydrogen bonds, guanine and cytosine links 
require three bonds each and needs higher energy to split. Also the surrounding envi-
ronment of the duplexes is important to achieve separation; in fact, chemical methods 
are also developed in order to reduce the thermal stability. An easier way to achieve de-
naturation is changing the pH of the environment to an alkaline solution; high concen-
10 
 
tration of hydrogen ions may be formed close to nucleotides, for capturing the hydrogen 
bond and splitting the duplex. Denaturation is a fundamental process frequently used as 
a step for molecular biology applications such as Polymerase Chain Reaction (PCR) and 
reclamation of probes in hybridization assays [5] [6].  
 
Fig. 1.2 - Example of a melting curve [4]. 
1.3 Biological assay 
Measurements are regularly needed in life-science to achieve qualitative and quantita-
tive information about the target or its functional activity with respect a target entity or 
its functional activity inside a biological environment. These experiments are known as 
biological assays and the target is called analyte. Several kinds of assays are employed 
depending on nature of the analyte, but they all have a few common principles. First of 
all, some pre-analysis processing makes a crude selection of entities of the same nature 
of the analyte, preparing them for the actual analysis. The first real step in an assay con-
sists in discriminating the target component from similar ones; this is the task of a selec-
tive probe that specifically identifies the analyte by its specific attributes. Once isolated, 
the target has to be revealed; for this reason a label is used to convert the amount of ana-
lyte in a detectable signal. The signal provided in this way is usually very weak, so it of-
ten requires amplification which is achieved with a biochemical approach or with an
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appropriate instrumentation. A detection system is then necessary to transform the sig-
nal in an interpretable output: this final step may be visual crude methods or by sophis-
ticated electronic systems [7]. 
1.4 Buffer solutions 
In sample preparation, the last step before the hybridization assay involves diluting the 
DNA samples in special solutions; a buffer is a solution often used in biochemistry, that 
resists a change in pH when small amounts of an acid or an alkali are added to it. They 
are commonly made by adding a weak acid or base to deionised water plus a salt of the 
same substance, an then adjusting pH with hydrochloric acid (HCl) or sodium hydrox-
ide (NaOH); this composition is used in order to keep the contaminant in minor concen-
tration [8]. For instance, HEPES buffer is a slightly basic buffer very often used in bio-
logical assays because it is resistant to carbon dioxide produced during cellular respira-
tion [9][10]. Specific buffers are used in hybridization to facilitate binding or denatura-
tion. In the nucleic acid structure, each bond with a ring of sugar makes the phosphate 
backbone negatively charged, so two strings experience a repulsive force that must be 
overcome to achieve duplex formation. A significant presence of positive charged ions 
shields the interaction between the backbones; this is easily achieved by dissolving a 
sodium or potassium salt inside the buffer in which hybridization is performed. Com-
mon hybridization buffers like Saline Sodium Citrate and Saline Sodium Phosphate-
ETDA are not the only buffers suitable for this purpose; Phosphate Buffered Saline 
(PBS) without EDTA and its variation SPSC are also widely used in biology and work-
ing as well. PBS recipe is here taken as an example: 137 mM of sodium chloride (NaCl) 
and 2.7 mM of potassium chloride (KCl) are the salts related to the weak bases which 
consist in 10 mM of sodium phosphate (Na
2
HPO
4
) and 1.8 mM of potassium phosphate 
(KH
2
PO
4
); the pH then reaches 7.4. Buffers are used to induce denaturation when the 
low stability of the membrane, probe or label does not permit it to be achieved thermal-
ly. This solution, rich in denaturant chemicals achieves the splitting of duplexes at room 
temperature: compounds with many H-bond groups compete with those in the nucleo-
bases and their presence may turn the melting point down to room temperature. Urea 
and formamide are molecules rich in amine groups, so they are good to this end. With 
some chemicals known as aldehydes, it is also possible to make covalent modification 
of nucleotides in order to avoid the Hydrogen bonds, splitting the duplexes [5]. Usual
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compositions of denaturant buffers are 3-6 moles of urea in a litre of deionised water or 
formamide at 30 to 80% of the volume (v/v) in PBS [11]. 
1.5 Selective probes 
The probe, in a hybridization assay, is a population of identified DNA strands with 
complementary sequence to target, labelled to detect and discriminate the target. Since 
hybridization is also allowed between different types of nucleic acid, probes may be of 
several kinds. To achieve simple DNA probes, double helixes are provided by cell-
based cloning or PCR and split by denaturation, but using both the strands will make the 
sensitivity substantially lower due to self-hybridization. Single strand probes may also 
be achieved by polymerization from another nucleic acid strand, reproducing the natural 
mechanism that occurs inside cells. Thus, with the help of enzymes, a process known as 
transcription gives RNA probes from complementary DNA, and backward reversal 
transcription allows direct generation of single DNA strands. Single strands of oligonu-
cleotides may also be made by chemical synthesis, shorter and more specific. Usually 
DNA probes are preferred because of the easy degradation of RNA by widely present 
enzymes named Ribonuclease (RNase) [6]. 
1.6 Achieving signal 
In assays, markers attached to biological entities are exploited to achieve a detectable 
signal from the analyte is the employing of. The method used to conjugate markers is 
called labelling and depends on the application; usually the most versatile approaches 
involve in vitro procedures during nucleic acid synthesis or adding a labelled group to 
its terminal nucleotide. Traditionally, labelling was conducted by incorporating radioac-
tive isotopes (often 
32
P, 
33
P, 
35
S or 
3
H) to produce an image on a photosensitive gel. The 
advantages of radioactive labelling include the emission of short wavelength β-particles 
which ensure high sensitivity; unfortunately it requires long exposure time and it pre-
sents ionizing radiation hazard. More recently, cytochemistry has developed non-
isotopic labels, classified by detection methods. Direct labelling consists in using a 
marker bonded on the string and ready to be detected. An optical approach using fluor-
ophores is usually preferred because it is better suited to detection with optical micros-
copy. Otherwise with indirect labelling, a reporter group is bonded on the nucleic acid; 
role of the reporter is to link an affinity molecule carrying the marker. Whereas direct