QUANTUM DOT BASED BIOSENSORS

A Seminar Report

Submitted by

AMAL KAMAL

in partial fulfillment for the award of the degree of

BACHELOR OF TECHNOLOGY

IN CHEMICAL ENGINEERING

At TKM College of Engineering

Department of Chemical Engineering, Kollam

JULY 2017

1
 TKM College of Engineering, Kollam 695005

Department of Chemical Engineering

CERTIFICATE

This is to certify that the project / dissertation entitled, QUANTUM DOT BASED
BIOSENSORS, which is being submitted herewith for the award of B.Tech., is the result of
the work completed by Amal kamal under my supervision and guidance within the four walls
of the institute and the same has not been submitted elsewhere for the award of any degree.

Prof Abdul Rasheed Prof Dr K B Radhakrishnan

Guide Head of Department

2
 DECLARATION

I hereby declare that the project/ dissertation entitled, QUANTUM DOT BASED
BIOSENSORS, was carried out and written by me/ us under the guidance of Prof Abdul
Rasheed, Professor, Department of Chemical Engineering,TKM College of Engineering . This
work has not been previously formed the basis for the award of any degree or diploma or
certificate nor has been submitted elsewhere for the award of any degree or diploma.

Place: Kollam Amal kamal

Date: July 2016 Roll No. 7612

3
 ACKNOWLEDGEMENT

I, Amal kamal, seventh semester student of the Chemical engineering department of TKMCE
, would like to express my sincere gratitude to my department for giving me such a wonderful
opportunity to present a seminar on the topic Quantum Dot Based Biosensors, which has led
to an increase in my knowledge and presentation skills , for which i am grateful. I would like
to thank Professor Abdul Rasheed for his invaluable guidance throughout this work. I would
like to thank our advisor Assistant Professor Saibi R for her guidance. I would also like to take
this oppurtunity to thank the HOD Dr.K.B.Radhakrishnan for his support and help.I also thank
all the staff members of the Chemical Engineering Department of TKMCE, my parents and my
peers for their help and support.

Amal Kamal

H7 7612

TKMCE

4
 ABSTRACT

Biosensors are one of the most important components of medical science and environmental
engineering among other aspects. Sensitive, effective and rapid detection of specific
biomolecules is of tantamount importance in diagnosis of medical conditions, biological
engineering and environmental protection. Quantum dots are essentially nanometre-scale
semiconductor crystals with unique optical properties that can be used in creating state of the
art biosensors and chemical sensors. Quantum dots typically have a very broad continues
absorption spectra, at wavelengths extending from the ultraviolet to the visible region,
depending upon their particle size. The broad excitation, narrow size yunable emission spectra,
high photochemical stability, resistance to photobleaching and alterations in Ph and
temperature, makes quantum dots an ideal candidate for the fabrication of various types of
biosensors. In this seminar, we go through various types of biosensors that are fabricated from
quantum dots and their applications in various fields.

5
 1. OVERVIEW BIOSENSORS AND QUANTUM DOTS

1.1 BIOSENSORS

The briefest of descriptions defines biosensors as an analytical device which converts a

biological response into an electric signal. The term biosensor covers a wide variety of
sensory devices used to determine the concentration of substances and other parameters of
biological interest even where they do not use a biological system directly.

The device is made up of a transducer and a biological element that may be an antibody, a
nucleic acid, or an enzyme. The biological elements interact with the analyte that is being tested
and the biological response hence obtained is converted into an electric signal by the transducer
and the output obtained is analysed. The vast majority of biosensors have immobilized
enzymes. The performance of the biosensor is mostly dependent on the specificity and
sensitivity of the biological reaction, besides the stability of the enzyme.

A biosensor has mainly two components :

1.A biological component, which consist of enzymes or cell

2. A physical component, which consists of the transducer and amplifier.

Figure 1 A diagrammatic representation of a biosensor

6
In a biosensor, the biomolecules are immobilized on the surface of the transducer, which also
act as the point of contact between the analyte and the transducer. The interaction between the
biomolecules and the analyte produces a physiochemical change in tyhe transducer surface.
This change is picked up by the surface and is converted to electric signals. These signals are
then made to undergo amplification and interpretation and finally displayed.

The essential features of a biosensor are ,

1. A biosensor should be highly specific, i.e., it should be specific to the molecule or

analyte that it interacts with.
2. The reaction should not be susceptible to change in parameters like Ph, temperature,
stirring etc, so that minimal pre treatment is needed for analysis.
3. The biosensor should be durable and able to withstand repeated usage.
4. The results obtained should be precise, stable and accurate and need to be free from
electrical noise.
5. A biosensor should be cheap and portable. It should be capable of being operated by a
semi-skilled operator.
6. If the biosensor is going to be used for the purpose of invasive monitoring, it should be
tiny and biocompatible. It should not be susceptible to proteolysis or fouling.

1.1.1 TYPES OF BIOSENSORS

According to the mode of interaction, biosensors have been classified into two categories;

Catalytic biosensor : In catalytic biosensors, the interaction of the analyte and the
biomaterial in the sensor results in the transformation of the analyte into a new chemical
molecule. Here the biomaterial used is mainly enzymes.

Affinity biosensor : In this type of biosensors, the analyte binds to the biomolecule upon
interaction. These sensors are mainly composed of nucleic acids and antibodies.

The main types of biosensors are :

1. Calorimetric Biosensor : Enzyme-analyte reactions which are exothermic in nature

releases heat and the subsequent change in temperature is detected by the transducer.

7
 2. Potentiometric Biosensor : Enzyme-analyte reaction produces an electric potential,
which is detected by the transducer.
3. Amperometric Biosensor : The enzyme and biological material reaction is a redox
reaction, which results in the movement of electrons , which is detected by the
transducer.

4. Optical Biosensors : The reaction between analyte and biological material absorbs or
releases light, which is detected by the transducer.

5. Acoustic Wave Biosensors : A change of biomass is experienced by the biological

material, which is detected by the transducer.

1.2.QUANTUM DOTS

Quantum dots are semiconductor crystal of nanometric dimensions.They have distinctive

conductive properties , largely determined by their size. These semiconductors are so small
that their electronic and optical properties differ largely from the properties of larger particles.
In quantum dots, the motion of excitons, which are bound pairs of valence band holes and
conduction band electrons, is confined to all three spatial directions.

Quantum dots can be made of materials such as silicon, indium arsenide, cadmium sulphide
orcadmium selenide. The size of quantum dots vary from 2nm to 10nm (diameter). The most
distincite property of quantum dots is fluorescence. It is this particular property of quantum
dots, which lead to its widespread applicatiopns ns and research. Fluoroscence is the property
by which the semiconductor nanoparticles emits certain colours when they are being
illuminated by light. When the semiconductor surface is subjected to radiation or light, some
of the electrons in its lattice attain enough energy to break free from the atom, which results in
the creation of a conduction band , in which the electrons are free to move around and hence
conduct electricity. When these excited electrons drop back into the valence band, they tend to
emit light and the colour of the emitted light depends on the difference in energy between the
valence band and the conduction band. The colour of the emitted light depends upon the size

8
of the nanoparticle. For example, for smaller nanoparticles, the energy difference between the
valence band and the conduction band will be high, resultin in the semiconductor emitting a
blue colour light. For larger nanoparticles, this energy difference between the conduction band
and energy band will be lower, resulting in emission of red colour.

Quantum dots are widely used in biosensors because they are resistant to photobleaching,
alterations in pH and temperature and to denaturants of biomolecules.

Fluoroscence is a very powerfull and important tool in biological research and applications and
its relevance relies greatly on the availability of sensitive and selective fluorescent probes [1].
Susceptibility to chemical changes in the medium, fixed emission spectra, photobleaching
etc.,are some of the main problems that plagued the earlier and usually used chemical
fluorophores. The spectroscopic and chemical features of the quantum dots makes them a very
good alternatives to traditional fluorescent probes in a variety of applications, including in
biosensors.

Figure 2. Variation of emitted light with size of quantum dots

Due to the simplicitry of crystallization and the availability of precursor, Cd-chalcogenide

nanocrystals have been the most favoured acolloidal quantum dot, in terms of research. Several
synthetic methods have been reported since the first synthesis of monodisperese
CdE(E=S,Se,Te) nanocrystals[2] and extensive reviews on the synthesis of nanocrystals can be
found [3]. Most of the time quantum dots are synthesised by using a coordination solvent,

9
which makes them soluble in organic solvents and prevents their agglomeration by serving as
a stabiliser. Since we are mainly discussing the biological applications of the quantum dots,
these crystals, which are hydrophobic in nature need to be transferred to the aqueous solution
using various methods like surface silanization, embedding in polymer shell etc. Therefore to
correctly understand their optical properties, an indepth knowledge of their surface chemistry
is necessary.

Most of the sensing assemblies that have been designed till now do not make use of quantum
dots as mere passive labels. Instead these systems are based on energy flow such as the transfer
of electronic excitation energy between the components of such nanoassemblies[4]. These
kinds of energy transfer can occur when light energy absorbed by the quantum dots is
transferred to a nearby acceptor species like an organic acceptor. This occurs via a process
called Fluorescence Resonance Energy Transfer (FRET). The rate at which the energy is
transferred between the donor and acceptor depends upon a lot of factors like their relative
orientation, spectral overlap, distance between them etc.

Figure 3 (a) emission spectra of CdSe/ZnS QD ; (b) illustratiion of size tunable QDs and creation of exciton

Another type of energy transfer called Bioluminescence Energy Transfer(BRET) is suitable for
luminescent quantum dots as it gets rid of the difficulties that arise while we use quantum dots

10
as acceptors. In this process, the photon generating process from a chemical reaction is used to
transfer the excitation energy nonradiatively to a proximal fluorescent acceptor. In comparison
to FRET, this process required the acceptor and donor to be in close proximity.

2. LITERATURE REVIEW

The surface chemistry of luminescent quantum dots has encouraged the development of various
probes. Representative examples of quantum dot based biosensors have been taken into account
in this study. Owing to the fact that the quantum dots have a vast array of fascinating properties,
the important applications are vast and some of these applications cannot be discussed in this
review.

Quantum dot based pH probes is an important area of discussion. By capping inorganic QDs
with organic ligands, we can obtain the pathway for obtaining hybrid nanoconjugates, which
in turn lead to the design of pH sensitive QDs with a wide variety of promising application [1].
Using this method the surface of CdSe/ZnS QDs was capped with an organic compound, which
included a dithiloane anchoring group, a 1,3-oxazine ring, an electron rich indole, a 4-
nitrophenylazophenoxy in their skeleton[2]. The basis of the pH sensing is the opening up of
the 1,2-oxazine ring upon the addition of base or acid. A different FRET based approach can
also be considered (between CdSe/ZnS QDs encapsulated within an ampiphilic polymer and a
pH sensitive dye conjugated on the cap surface) . Mercaptosuccinic acid-CdSe/ZnS QDs can
also be used for the detection of urea based on the pH sensing abilities of the earlier mentioned
QD [3].

Detection of biomolecules like nucleic acids is another important area of application of QDs.
A wide variety of methods for DNA sensing has been developed having reduced levels of
interfering molecules and good selectivity. Mainly two concepts were designed for DNA
sensing based on the specificity of hybridisation between target-probe olegonucleotides [4]. In
both these methods, the coincidences of two different wavelength fluorescent signals emitted

11
from the QD/DNA target probe lead to the detection of targets [5]. There has also been studies
on the hybridisation of DNA and cleavage processes The treatment of QD/dye-DNA structure
with deoxyribonuclease cleaved the DNA duplex and lead to the restoration of fluoroscence
properties, while the hybridisation was done by following FRET between QDs and a molecular
fluorophore. The dynamics of DNA telemerisation and replication have also been studied [6].
As telemerisation proceeded, the fluorescence emission of the QDs decreased, with an
associated increase in the characteristic emission of the fluorophore by FRET from the Qd to
the dye molecules incorporated into the telomeric units. There are also FRET based DNA
hybridisation sensors that do not require covalent immobilisation of the probe molecules,
thereby minimising the DNA modification. Exploiting QD technology to improve the detection
of interaction between DNA and anticancer drugs is of very high interest, from biomedical
point of view. In RNA-peptide interaction studies, FRET mechanism between organic dyes
and QD have been successfully employed. The possible applications of peptide capped QD
include monitoring alterations of enzymatic activities , which has direct link to important
biological processes and diseases. The capping chemistry with DNA aptamers can also help in
providing selective binding to a protein target.

Real time detection of hydrogen peroxide 2O2 is another important application of QD based
biosensors. It is important in the sense that it helps in the study of biological phenomenon and
oxidative stress related diseases. All this being said, selective detection and accurate
monitoring of H2O2 is still a challenge due its low concentration, high reactivity and high
diffusivity. A biomimetic enzyme, ultrasmall Fe3O4 decorated on a three-dimensional
graphene nanocomposites was used for this process [7]. This enzyme free biosensor was used
to detect hydrogen peroxide from living cells by controlling cell numbers and stimulation drug
dose. Various types of biomimetic enzymes with different components have been used as
catalysts for the enzyme free biosensor. Noble metals exhibit the best activity, but their high
cost and harsh synthesis methods greatly limit their usage. There are various other feasible
alternatives such as cobalt, manganese oxide and titanium dioxide. Ferric chloride
hexahydrate, hydrogen peroxide, sodium bicarbonate, glucose, Phorbol 12-myristate-13-
acetate, ascorbic acid ,uric acid, dopamine hydrochloride , potassium superoxide and natural
graphite were the main reagents used for the fabrication of the graphene nanocomposites and
these nanocomposites were used for the real time detection of hydrogen peroxide from living
cells. The sensor that has been designed offers excellent conductivity and catalyst durability
with higher sensitivity and selectivity, fast response and low detection time.

12
Pesticides are still in widespread use among in the agricultural industry , dspite the fact that
they cause a wide variety of environmental and health problems, which is a direct result of high
levels of accumulation of these compounds. Here, QD based biosensor was used to detct and
quantify the herbicide 2,4-dichlorophenoxyacetic acid. The quantitative analysis of 2,4-
dichlorophenoxyacetic acid was carried out by a fluroimmunoassay biosensor [8]. The ever
expanding research in the field of nanomaterials and biosensors lead to the development of
QD- based sensors that is capable of detecting analytes resulting from industrial or warfare
applications. The fluorescent determination of various polycyclic aromatic hydrocarbons can
be carried out using QDs with cycodextrins or cyclic oligosaccharides.

Sensing of ions via analyte induced changes in photoluminescence of quantum dots is a very
active and thriving research fields[9].Different capping agents like L-carnitine has been used
to determine the usefulness of QDs as ion probes [10]. In general, the interaction of ions with
quantum dots resulted in a fluroscence quenching, which can be attributed electron transfer
processes, the nonradiative recombinations pathways and the inner-filter effects. A sensor for
Hg2+ was developed based on a selective fluorescence quenching of CdSe/ZnS quantum dots
modified with sulphur calixarenes [11]. Anothe example of am ion detector was the FRET
based system developed between TGA-CdTe quantum dots and acceptor butyl-rhodamine B in
the presence of a surfactant, to increase the efficiency of FRET. Recently, using
azamacrocycles, a selective nanosensor based on PET fluorophore-ligand have been designd
for the sensing of Zn2+ ions. These ion sensitive quantum dots exhibited good selectivity to
Zn2+ in comparison to other cations and hence can be applied for the determination of zinc in
physiological media. When compared to the usage of quantum dot based sensors for detection
of cations, the usage of the same for anion detection is practically unheard off. The selective
sensing of multiple ions simultaneously can be enhanced by modification of the quantum dot
surface.

3.QUANTUM DOT BASED pH PROBES

13
The identification of pathways to obtain hybrid nanoconjugates by capping inorganic quantum
dots with organic ligands lead us to the creation of pH sensitive quantum dots with very
promising applications for a variety of analytical purposes, particularly for the development of
luminescent chemosensors[16]. Using the above mentioned strategy, tyhe CdSe/ZnS surface
was capped with an organic compound incorporating a dithiolane anchoring group, a 4-
nitrophenylazophenoxy chromophore an electron rich indole and a 1,3-oxazine ring in their
skeleton [17]. The 1,3-oxazine ring opens up on the addition of an acide or base and this is
principle upon which pH sensing is based. In an acidic pH , the opening up of the ring results
in 4-nitroxyphenylazophenol, which does not absorb on the in the visible region, which means
that the chromophore does not accept excitation energy from the quantum, dots and is also a
poor donor of electrons. But in basic pH , the chemical stimulation generates a 4-
nitrophenylazophenolatr chromophores and is trasnduced into luminescence quenching due to
PET from quantum dots to the chromophores adsorbed on their surface.

The CdSe/ZnS nanocrystal can also be used to create a ratiometric pH sensor. It is a well known
fact that nanocrystals are usefull in identifying locations in a microenvironment, their inherent
insensitivity to the presence of most biological or chemical agents renders them practically
useless or of limited utility in certain cases. The recently developed water soluble CdSe
nanocrystals with the ability to sense target analytes irreversibly using FRET as a single
transduction system[18]. With this study, it has been shown that a reversible quantum dot based
fluoroscent sensor can be designed by conjugating a dye with an equilibrium response to an
analyte to the surface of a CdSe/ZnS quantum dot, and hence it has been shown that a general
method for the development of ratiometric sensors can be developed by properlly controlling
the energy transfer between the nanocrystal and dye.

For creating the above mentioned sensor, high quality CdSe nanocrystals overcoated with with
zinc sulphide adn capped with trioctylposphine oxide ligands were encapsulated by a
hydrophobically modified polyacrylic acid (see figure 4).The squaraine dye shown in figure 4
was linked to the the backbone of the polymer via ester linkages using 1-ethyl-3-(3-
dimethylaminopropyl)carbamide as the coupling agent. It has also been found that the same
dye conjugation method can be applied to water soluble nanocrystals that have been cap
exchanged with thio-functionalizedpolyethylene glycol. To ensure that the unreacted dyes have
been removed from the sample, the sample can be easily made to undergo purification using

14
dialysis filter. By modulation of the FRET efficiency arising from the engineered overlap of
the pH sensitive dye absorption spectrum with the quantum dot emission, the sensing action of
the nanocrystal squaraine conjugate is imparted. When pH is lowered, the spectral overlap
between the nanocrystal and the dye absorption increases, due to the pH dependence of the dye
absorption spectrum. At increased pH , since the spectral overlap is small, the FRET should be
inefficient. Therefore , the emission spectrum should be dominated by luminescence from the
nanocrystal. The modulation of the FRET efficiency by pH results in an emission dependence
between the nanocrystal and the dye that is naturally ratiometric[19]. The ratiometric approach
to pH sensing is much more powerfull when compared to typical chemo- and biosensors which
displays a single intensity based response to analytes (ie., either darkening or brightening) since
the ratiometric construct is not sensitive to collection efficiency or fluctuations in of light
excitation as sensing is self referencing.

Figure 4. A sensor constructed from a colloidal CdSe NC that is overcoated with an outer layer of ZnS

There are also research going on in the basis of thioalkyl-functionalized quantum dots, which
are pH sensitive[20],which opens up the door to a wide variety of applications. In this aspect,
mercaptic acid-CdSe/ZnS quantum dots have been used as an intracellular pH sensor by
observing a quenching of quantum dots fluorescence in acidic pH [21]. This strategy results in
the quantum dot being much more resistant against photobleaching. Urea detection is possible
by using the pH sensing capabilities of mercaptosuccinic acid-CdSe/ZnS quantum dot, in an
environment which also contains urease enzyme [22]. Hydroxide anions are generated by the
urease catalyzed hydrolysis of urea, which gradually increased the pH value of the solution,

15
which in turn leads to an enhancement of quantum dots fluorescence that could be correlated
with increasing concentration of urea. These studies are revealing of a long path for future
application of quantum dots as pH probes.

4. DETECTION OF NUCLEIC ACIDS

For the detection nucleic acids, various DNA sensing approaches have been developed. These
techniques are characterised by reduced levels of interfering molecules and good selectivity.
For instances, two concepts were designed based on the specificity of hybridisation between
target-probe oligonucleotides[25]. In the first approach, two single stranded DNA probes, one
biotinylated (modified via a biotin-streptavidin interaction) , and the other conjugated to an
organic fluorophores, were mixed with the target DNA to form a sandwich hybridisation
structure, which was captured by the streptavidin modified quantum dot. The organic
fluorophore-labelled structures coupled to the quantum dot serves dual purposes; it functions
as a fluorescent tag and the quantum dot also worked as nanoscaffolds, resulting in amplified
signals. In the second approach, two quantum dot having different emission wavelengths, were
modified with two single stranded DNA probes by a biotin-streptavidine interaction. These
modified DNA probes were designed in such a way that it will bind to two different binding
sites in the same target DNA strand, so that cross-linking of the quantum dots will occur on
target-probe hybridisation.

Unlike the above mentioned biosensors, there are also FRET based DNA hybridisation sensors
that minimizes the modification the DNSA strand. Water soluble quantum dots were prepared
using thioglycolic acid (TGA) as the capping ligand, and this negatively charged quantum dot
was further incorporated in a cationic polymer solution of polydiallyldimethylammonium as
the electrostatic linker. The positively charged quantum dot that was obtained, acted as a FRET
donors to dye acceptor labelled single stranded DNA.

16
 Figure 5.Illustration of various QD based sensing configuration for nucleic acids

The existing quantum dot technology can be utilised to improve the detection of the interaction
between DNA and anticancer drugs. In a recent study, a model anthraquinone anticancer drug
(mitoxantrone, MTX), demonstrated the usefulness of PET based sensors to study the
interaction between MTX and DNA. The photoluminescence of the quantum dot was quenched

17
by the MTX adsorbed on it, through an electron transfer process from the quantum dot to MTX,
which could be restored in the presence of DNA that bound MTX and removed it from the
surface of the quantum dot [26]. With this technique, the need for immobilizing processes,
modifying or labelling was removed and good sensitivity of detection was achieved.

5. REAL TIME DETECTION OF HYDROGEN PEROXIDE

Reactive oxygen species (ROS) are a family of bio regulatory and signaling molecules that are
continuously generated, transformed and consumed in the biological system by means of both
physiological and pathophysiological pathways. And they are considered as the small
molecules responsible for the mediation of redox modification of cell biology and pathology
[23]. One of the most stable representatives of the ROS species is hydrogen peroxide, generated
in normal metabolism as signalling molecule for growth factors, second messengers and signal
transduction. Studies however, has shown that after undergoing abiotic and biotic stresses like
drug stimulation, UV light and pathogens, the amount of hydrogen peroxide in the cells can
increase drastically, furthermore, hydrogen peroxide can penetrate freely into almost any
cellular compartments through its membrane. Increased concentration of hydrogen peroxide in
cells can directly lead to damages in lipids, DNA and proteins, as well as some disease ranging
from cancer to neurodegenerative diseases. Therefore it is clear that the accurate detection and
monitoring of hydrogen peroxide in cells is really important.

For the in situ detection of 2 2 a facile synthetic 3 4 quantum dot supported by a three
dimensional graphene (3DG) network was synthesised and used.3 4 has intrinsic peroxidise
like activity similar to peroxidise enzyme that contains ferric and ferrous ions in their reaction
centres. 3DG has been formed by self assembling 2D graphene sheets, maintains a porous,
flexible and 3D structure with high specific surface, fast charge mobility and excellent
conductivity. When these nanocomposites were used as a catalyst for 2 2 detection, it
delivered ecxcellent sensitivity, selectivity and faster amperometric response. More
importantly, enzyme free biosensors are cost effective and can be successfully used for the
detection of trace amounts of 2 2 released by living tumor cells.

18
REAGENTS AND MATERIALS USED :

Ferric chloride hexahydrate (FeCl36H2O), Sodium Bicarbonate (NaHCO3),

hydrogen peroxide (H2O2, 30%) , glucose, Phorbol 12-myristate-13-acetate (PMA, 99.5%),
catalase (cat, 2000-5000U/mg), L-ascorbic acid (AA), uric acid (UA), dopamine
hydrochloride (DA), potassium superoxide (KO2) and natural graphite. All other reagents are
of analytical grade and used as received without further purification. Deionized (DI, 18 M)
water was also used in all the experiments.

PREPARATION OF /3DG NANOCRYSTALS :

Graphene oxide (GO) was prepared from natural graphite powder by a modified
Hummer's method. Then, an aqueous suspension of GO at a concentration of 2.5
mg mL-1 was prepared to sealed in a Teflon-lined autoclave and heated at 180 C for
12 h. Next, the obtained 3DG hydrogel was washed with DI water several times
and freeze dried in vacuum environment. The Fe3O4 QDs were prepared to decorate
on the 3DG networks via in situ self-assembly by hydrothermal treatment. In a typical
synthesis, appropriate amount of freeze-dried 3DG was dispersed in 30 mL DI water
under sonication. 0.7095 g FeCl36H2O and 0.6616 g NaHCO3 were added
accordingly into the homogeneous 3DG aqueous dispersion with vigorously stirring
for 30 min. After adding 0.088 g of L-ascorbic acid, the mixture was further stirred
for another 10 min before transferred to Teflon-lined autoclave (50 mL) and then
heated at 150 C for 6 h in an air-flow electric oven. Subsequently, the obtained black
precipitates were separated by magnet to remove the adsorbent, washed several times
with DI water and freeze-dried, the Fe3O4/3DG NCs were obtained. In order to
evaluate the effect of different loading capacity of Fe3O4 QDs in this hybrid on H2O2
detection, the mass ratio of 3DG to iron oxide could be controlled by changing
feedstock concentration of 3DG dispersion in separate experiments, and the pure
Fe3O4 QDs were also synthesized under the same experimental conditions without
the3DG.

The enzyme free biosensor that was prepared using the above mentioned method offers the
unique electrochemical property of excellent conductivity, low detection time, selectivity,
sensitivity and catalyst durability with fast response. Furthhermore, the nanoscale flexible
graphene composites are promising for constructing a miniaturized probe to realise the

19
miniaturisation of biosensors [24]. The work that we have discussed above shows us that the
biosensor that was fabricated exhibits excellent ability to accurately detect the amount of 2 2
released from living cells by controlling cell numbers and stimulation drug dose. These studies
indicates a strong future for Fe3 O4 /3DG nanocrystals in pathological investigations, drug
discovery and further fundamental research.

6. DETECTION OF ORGANIC COMPOUNDS

Pesticides and herbicides are still widely used in many households and farms, despite the fact
that usage in high levels can cause a variety of environmental and health problems, the
accumulation of these xenobiotic compounds in the biosphere can lead to a variety of problems
like ground water contamination etc., and can also lead to a wide variety of disease and other
health problems. Quantum dots and immunological techniques were combined to detect and
quantify the the herbicide 2,4-dichlorophenoxyacetic acid. The enzyme alkaline phosphate
was conjugated to the herbicide 2,4-dichlorophenoxyacetic acid, followed by the formation of
an amide bond between the carboxylic group of the MPA-CdTe quantum dot and the amine
group of alkaline phosphatise. Based on a fluoroimmunoassay biosensor, the quantitative
analysis of 2,4-dichlorophenoxyacetic acid was then carried out.

The sensitivity of quantum dots to hydrogen peroxide (2 2) lead to development of versatile

fluorescent based quantum dot based sensors, which can potentially be applied to a number of
oxidased that biocatalyses the generation of 2 2 . The inhibition of acetylcholinesterase
(AChE) by the carbamate neostigmine exemplified the proposed system. In this hybrid method
AChE was responsible for hydrolysing acetylcholine to choline and choline oxidase
subsequently oxidised choline to betaine with the concomitant generation of H2O2 and
quenching of the fluorescence of CdSe/ZnS QDs (Figure 6). In the presence of the inhibitor,

20
the biocatalytic cascade was interrupted and QDs fluorescence was not quenched

Figure 6. QD based sensor for AChE pesticide

MPA-CdSe/ZnS electrostatically conjugated to an organophosphorous hydrolase was used to

detect paraoxon, which quenched the photoluminescence of the QDs probably due to a change
in the secondary structure of the enzyme upon pesticide binding [27].

The intense research going on the field of quantum dots and their surface chemistry lead to the
development of quanyum dot based biosensors for the detection of analytes resulting from
industrial or warfare applications. Quantum dots fluorescence was modulated in the presence
of nitroaromatic compounds or diamines, providing new pathways to assess these and related
compounds. The conjugation of quantum dots to histidine tail engineered proteins has been
applied in self-assembly of a recombinant specific antibody of the explosive 2,4,6-
trinitrotoluene (TNT) onto QDs surfacehas lead to the detection of this compound by
competitive immunoassay. The fluorescence quenching of QDs by various detergents of
common use showed the role of the surface charge in the quenching process and has been
explained on the basis of nonradiative recombination with deep-traps, less mobile holes and by
stabilizing electrons in surface traps [28]. The applicability of TGA-CdTe QDs for the
determination of cationic surfactants in real samples was satisfactory, showing good sensitivity
and selectivity [29]. Functionalized QDs with cyclodextrins, cyclic oligosaccharides with a
variable-sized hydrophobic internal cavity for the formation of inclusion complexes and a
hydrophilic external surface to retain the aqueous solubility of the macrocycle, have been

21
proposed as selective probes for fluorescent determination of various polycyclic aromatic
hydrocarbons [30].

ION SENSING

The sensing of ions by inducing changes in the photolumiscene of quantum dots is a very
thriving research field and many thiol based capping strategies have emerged, which include
the use of a number of thioalkyl acid ligands (thioglycolic acid,MMA and TGA; dyhydrolipoic
acid, DHLA). The carboxyl groups presdent gets exposed to the surrounding aqueous solution
as the self assembly proceeds via metal-thiol affinity interactions. Therefore the ligand is
responsible for both the solubility of quantum dot in aqueous environment and for participating
in response to metal ions. Different capping agents like L-carnitine or bovine have been used
to portray the usefulness of quantum dots as ion probes. Generally speaking, a fluoroscene
quenching is induced in the quantum dot due to the interaction of the quantum dot with the
ions. This can be attributed to nonradiative recombination pathways, inner-filter effects and
electron transfer processes, although fluorescence enhancement can also be observed and
passivation of trap statesorcdefects on the surface of the quantum dot is usually the mechanism
ascribed for the increase in quantum yield that has been observed [31]. Surface ligands play a
pivotal role in the fluorescence response of quantum dots to metal ions and they play a
significant role in ion selectivity.

A zinc ion selective nanosensor based on PET fluorophore-ligand have been designed using
azamacrocycles, a class of nitrogen containing analogues to crown ethers, conjugated via an
amide link to MPA-CdSe/ZnS quantum dot [32]. The radiative recombination process was
disrupted due to the interaction of azamacrocycles with the photoinduced quanbtum dot charge
carriers, mostly because the transfer of hole from the quantum dot to azamacrocycles was
stabilized by the lone pair of electrons of the nitrogen atom. The fluorescence intensity was
recovered when the azacrown was penetrated by the zinc ion, requiring the involvement of
nitrogen atom in the coordination, which became unavailable for participating in the charge
separation process. These ion sensitive quantum dots based showed good selectivity to zinc
when compared to other cations and their applications in zinc detection was shown.

22
There are sensors available for the detection of Hg2+ ions. These sensors have been developed
based on a selective fluoroscene quenching of CdSe/ZnS quantum dot modified with sulphur
calixarenes [33]. For this system, the influence of other metal ions were very weak and the
system showed a detection limit in the nm range.

Comparing to the use of quantum dot based sensors for the detection of cations, the usage of
the same for the detection of anions has been almost unexplored. For example, CdSe QDs have
been functionalized with 2-mercaptoethane sulfonate for optical detection of cyanide ions due
to quenching of the fluorescence, reaching a limit of detection in the M range [34]. More
recently, a platform based on PET has been proposed for the anion detection using CdSe/ZnS
quantum dot capped with a charge neutral thiourea receptor.

Some works have also demonstrated that by modifying the surface of the quantum dot, the
selective sensing of multiple ions simultaneously can be increased. Fluorescence quenching
was induced in aqueous media of the pentapeptide ( Gly-His-Leu-Leu-Cys) , which responded
with high selectivity to Ag+ and Cu2+ ions. Triethanolamine capped CdSe quantum dot
exhibited that it was responsive through an effective fluorescence quenching only when it was
associated with Hg2+ and I-. It was hypothesized that I bridged the binding between QDs and
Hg2+, allowing the formation of a stable complex and an effective electron transfer from the
QDs to the cation and consequent fluorescence quenching [35]. A recent work has revealed
that CdSe/ZnS quantum dot conjugates with an organic receptor, which allowed the selective
chromogenic detection of Cu2+ and Fe3+ ions simultaneously in a semi-aqueous solution.

CONCLUSION
23
From all of the applications that we have seen above, it is very clear that quantum dots have
extraordinary electronic and optical properties that have been studied for over a decade ,
making them one of the most prolific nanomaterial known to science. These semiconductor
nanocrystal offer a wide range of applications which include their usage as an active component
in nanostructure-biomolecule complexes, as well as them being used as a new type of
fluorescent probes (which we have gone through in this report).

A new strategy for biological and chemical sensing have been developed by tethering
emissive water solubilised nanocrystals to environmentally sensitive dye molecules. The
modulation of FRET efficiency between the emissive nanocrystal and the dye conjugated to
the nanocrystal surface lead to a ratiometric response to pH. the narrow , size tunable emission
spectrum of the nanocrystal enables to be FRET donors that may easily be custom engineered
to match the acceptor absorption feature of a dye conjugated to the surface of the nanocrystal,
hence this approach is general as a sensing construct. The ratiometric approach presented here
in addition to the photostability and broad excitation spectrum makes the quantum dots a very
versatile agent for biological and chemical sensing.

The enzyme free biosensor from living cells offers the unique electrochemical catalysis
properties of excellent conductivity, low detection time, higher sensitivity and selectivity and
catalyst durability with fast response. It is also clear from our study that the nanoscale flexible
graphene composites can be used in the miniaturisation of biosensors. By controlling cell
numbers and stimulation drug dose, the biosensor shows excellent ability for the in situ
detection of hydrogen peroxide. Hence, it is clear that the 3 4/3DG nanocrystals can be used
in further fundamental research and in pathological investigations.

For successfully sensing a wide range of molecules, various optical transduction schemes have
already been explored, allowing reduced interference of other compounds and low levels of
detection. Currently a reality, multiplex detection is an ever expanding research tool for
quantum dots in optical arrays. The major limitation to the development of reusable sensors is
the fact that majority of applications occur in bulk solutions and due to their known high levels
of toxicity, routine analytical applications are moving at a small pace. Accompanying the
continues progress in the synthesis and conjugation methods for increased stability and
sensitivity, these will undoubtedly be two of the challenges that ought to be tackled in future
studies.

REFERENCES

24
[1] Manuela F. Frasco ; Nikos Chaniotakis. Semiconductor Quantum Dots in Chemical
Sensors and Biosensors . Sensors 2009, 9, 7266-7286

[2] Murray, C.B.; Norris, D.J.; Bawendi, M.G. Synthesis and characterization of nearly
monodisperse CdE (E = sulphur, selenium, tellurium) semiconductor nanocrystallites. J. Am.
Chem. Soc. 1993, 115, 87068715.

[3] Park, J.; Joo, J.; Kwon, S.G.; Jang, Y.; Hyeon, T. Synthesis of monodisperse spherical
nanocrystals. Angew. Chem. Int. Ed. 2007, 46, 46304660.

[4] Manuela F. Frasco ; Nikos Chaniotakis. Semiconductor Quantum Dots in Chemical

Sensors and Biosensors . Sensors 2009, 9, 7266-7286

[5] Manuela F. Frasco ; Nikos Chaniotakis. Semiconductor Quantum Dots in Chemical

Sensors and Biosensors . Sensors 2009, 9, 7266-7286

[6] Tomasulo, M.; Yildiz, I.; Kaanumalle, S.L.; Raymo, F.M. pH-sensitive ligand for
luminescent quantum dots. Langmuir 2006, 22, 1028410290.

[7] Li, H.; Zhang, Y.; Wang, X.; Gao, Z. A luminescent nanosensor for Hg(II) based on
functionalized CdSe/ZnS quantum dots. Microchim. Acta 2008, 160, 119123.

[8] Yeh, H.-C.; Ho, Y.-P.; Wang, T.-H. Quantum dot-mediated biosensing assays for specific
nucleic acid detection. Nanomed.: Nanotechnol. Biol. Med. 2005, 1, 115121.

[9] Manuela F. Frasco ; Nikos Chaniotakis. Semiconductor Quantum Dots in Chemical

Sensors and Biosensors . Sensors 2009, 9, 7266-7286

[10] Patolsky, F.; Gill, R.; Weizmann, Y.; Mokari, T.; Banin, U.; Willner, I. Lighting-up the
dynamics of telomerization and DNA replication by CdSe-ZnS quantum dots. J. Am. Chem.
Soc. 2003, 125, 1391813919.

[11] Yanan Zhao; Danqun Huo; Jing Bao; Mei Yang,

Mei Chen,;Jingzhou Hou; Huanbao Fa; Changjun Hou. Biosensor based on
3D graphene-supported Fe3O4 quantum dots as biomimetic enzyme for in situ
detection of H2O2 released from living cells, Sensors and Actuators B:

[12] Vinayaka, A.C.; Basheer, S.; Thakur, M.S. Bioconjugation of CdTe quantum dot for the
detection of 2,4-dichlorophenoxyacetic acid by competitive fluoroimmunoassay based
biosensor. Biosens. Bioelectron. 2009, 24, 16151620.

[13] Manuela F. Frasco ; Nikos Chaniotakis. Semiconductor Quantum Dots in Chemical

Sensors and Biosensors . Sensors 2009, 9, 7266-7286

[14] Xie, H.-Y.; Liang, J.-G.; Zhang, Z.-L.; Liu, Y.; He, Z.-K.; Pang, D.-W. Luminescent
CdSe-ZnS quantum dots as selective Cu2+ probe. Spectrochim. Acta A 2004, 60, 25272530.

25
[15] Li, H.; Zhang, Y.; Wang, X.; Xiong, D.; Bai, Y. Calixarene capped quantum dots as
luminescent probes for Hg2+ ions. Mater. Lett. 2007, 61, 14741477.

[16] Manuela F. Frasco ; Nikos Chaniotakis. Semiconductor Quantum Dots in Chemical

Sensors and Biosensors . Sensors 2009, 9, 7266-7286

[17] Tomasulo, M.; Yildiz, I.; Kaanumalle, S.L.; Raymo, F.M. pH-sensitive ligand for
luminescent quantum dots. Langmuir 2006, 22, 1028410290.

[18] Willard, D.M.; Mutschler, T.; Yu, M.; Jung, J.; Van Orden, A. Directing energy flow
through quantum dots: Towards nanoscale sensing. Anal. Bioanal. Chem. 2006, 384, 564
571.

[19] Willard, D.M.; Mutschler, T.; Yu, M.; Jung, J.; Van Orden, A. Directing energy flow
through quantum dots: Towards nanoscale sensing. Anal. Bioanal. Chem. 2006, 384, 564
571.

[20] Susha, A.S.; Javier, A.M.; Parak, W.J.; Rogach, A.L. Luminescent CdTe nanocrystals as
ion probes and pH sensors in aqueous solutions. Colloid. Surf. A 2006, 281, 4043.

[21] Liu, Y.-S.; Sun, Y.; Vernier, P.T.; Liang, C.-H.; Chong, S.Y.C.; Gundersen, M.A. pH-
sensitive photoluminescence of CdSe/ZnSe/ZnS quantum dots in human ovarian cancer cells.
J. Phys. Chem. C 2007, 111, 28722878.

[22] Huang, C.-P.; Li, Y.-K.; Chen, T.-M. A highly sensitive system for urea detection by
using CdSe/ZnS core-shell quantum dots. Biosens. Bioelectron. 2007, 22, 18351838.

[23] B.C. Dickinson, C.J. Chang, Chemistry and biology of reactive oxygen species in
signaling or
stress responses, Nat. Chem. Biol. 7 (2011) 504-511

[24] Yanan Zhao, Danqun Huo, Jing Bao, Mei Yang,

Mei Chen, Jingzhou Hou, Huanbao Fa, Changjun Hou, Biosensor based on
3D graphene-supported Fe3O4 quantum dots as biomimetic enzyme for in situ
detection of H2O2 released from living cells, Sensors and Actuators B: Chemical

[25] Yeh, H.-C.; Ho, Y.-P.; Wang, T.-H. Quantum dot-mediated biosensing assays for
specific nucleic acid detection. Nanomed.: Nanotechnol. Biol. Med. 2005, 1, 115121.

[26] Yuan, J.; Guo, W.; Yang, X.; Wang, E. Anticancer drug DNA interactions measured
using a photoinduced electron-transfer mechanism based on luminescent quantum dots. Anal.
Chem. 2009, 81, 362368

[27] Ji, X.; Zheng, J.; Xu, J.; Rastogi, V.K.; Cheng, T.-C.; DeFrank, J.J.; Leblanc, R.M.
(CdSe)ZnS quantum dots and organophosphorus hydrolase bioconjugate as biosensors for
detection of paraoxon. J. Phys. Chem. B 2005, 109, 37933799.

26
[28] Hamity, M.; Lema, R.H.; Suchetti, C.A. The effect of tetraalkylammonium and alkyl
sulphate salts on the fluorescence bands of quantum-sized CdS. J. Photochem. Photobiol. A:
Chem. 1998, 115, 163168.

[29] Diao, X.-L.; Xia, Y.-S.; Zhang, T.-L.; Li, Y.; Zhu, C.-Q. Fluorescence-detecting cationic
surfactants using luminescent CdTe quantum dots as probes. Anal. Bioanal. Chem. 2007, 388,
11911197

[30] Diao, X.-L.; Xia, Y.-S.; Zhang, T.-L.; Li, Y.; Zhu, C.-Q. Fluorescence-detecting cationic
surfactants using luminescent CdTe quantum dots as probes. Anal. Bioanal. Chem. 2007, 388,
11911197

[31]Huang, C.-P.; Li, Y.-K.; Chen, T.-M. A highly sensitive system for urea detection by using
CdSe/ZnS core-shell quantum dots. Biosens. Bioelectron. 2007, 22, 18351838.

[32] Ruedas-Rama, M.J.; Hall, E.A.H. Azamacrocycle activated quantum dot for zinc ion
detection. Anal. Chem. 2008, 80, 82608268.

[33] Li, H.; Zhang, Y.; Wang, X.; Xiong, D.; Bai, Y. Calixarene capped quantum dots as
luminescent probes for Hg2+ ions. Mater. Lett. 2007, 61, 14741477.

[34] Jin, W.J.; Fernndez-Argelles, M.T.; Costa-Fernndez, J.M.; Pereiro, R.; Sanz-Medel, A.
Photoactivated luminescent CdSe quantum dots as sensitive cyanide probes in aqueous solutions.
Chem. Commun. 2005, 883885.

[35] Manuela F. Frasco ; Nikos Chaniotakis. Semiconductor Quantum Dots in Chemical

Sensors and Biosensors . Sensors 2009, 9, 7266-7286

27
\

28