Application of Nanoparticles to the Analysis of Biomarkers

iv 
ABSTRACT 
Nanotechnology is any technology which studies and exploits phenomena and materials that 
only occur at the scale of single atoms and small molecules: the nanometer scale. At this small 
scale, nanomaterials display unique physical and chemical features that lead to new properties 
depending on the size. When it decreases, in fact, the surface/volume ratio increases 
considerably, giving nanostructure unique optical, mechanical, photochemical, electronic and 
magnetic properties not seen at the bulk scale, which makes them attractive for a wide range 
of applications, such as environmental monitoring, detection of pathogens, proteomics, 
genomics, drug delivery, catalysis and bioanalysis. 
Within nanomaterials, nanoparticles have received great interest due to their attractive 
electronic, optical, thermal, catalytic properties and potential application in the fields of 
physics, chemistry, biology, medicine, and material sciences. Nanoparticles are clusters of a 
few hundred to a few thousand atoms sized between 1 and 100 nanometers, that behave as 
whole units in terms of transport and properties. They can be composed of a single constituent 
material (metal, semiconductor or oxide) or be a composite of several materials and can be 
realized in a variety of shapes, morphologies and phases. 
“Biomarker” can be defined as a characteristic that is objectively measured and evaluated as 
an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses 
to a therapeutic intervention. As the definition suggests, there are many types of biomarkers, 
including biochemical markers, physiologic markers, anatomic markers, histological markers 
and physical markers. They can be used in clinical practice to identify risk for or diagnose a 
disease, assess disease severity or progression, predict prognosis, or guide treatment. Tumor 
markers are substances, most often proteins, that can be found in the blood or urine when 
cancer is present. They can be products of the cancer cells themselves, or made by the body in 
response to cancer or other conditions. 
v 
What links biomarker analysis to nanotechnology is the possibility to modify and improve the 
standard analytical methods by the introduction of nanosystems acting for example as carriers 
for molecules, tracers to be detected, electron transfer mediators or catalysts of 
electrochemical reactions. 
In this work the attention has been focused on the analysis of an important tumor marker, 
protein CA 15-3, essential to monitor breast cancer treatments. 
Breast cancer is the most common cancer diagnosed in women. More than 1 million women 
worldwide are diagnosed with breast cancer each year. CA 15-3 is a glycoprotein mainly used 
to watch patients with breast cancer. In fact, elevated blood levels are found in less than 10% 
of patients with early disease and in about 70% of patients with advanced disease. The normal 
level is usually less than 30 U/mL (units/milliliter), but levels as high as 100 U/mL can 
sometimes be seen in women who do not have cancer. The CA 15-3 test is performed to aid 
physicians in the monitoring of treatment of women with invasive breast malignancies. 
It can also be used to diagnose recurrence of the disease following first-line therapy. By using 
the measurements obtained from the CA 15-3 test the physicians can help to determine if the 
patient is responding to treatment or if additional treatment or testing is required. 
The Enzyme-Linked Immunosorbent Assay (ELISA) is the technique most commonly used 
by molecular biologists for biomarker detection and quantification. ELISA tests are based on 
the principle of a solid phase enzyme-linked immunosorbent assay using the spectroscopic 
detection of a colored reagent. The assay system utilizes a monoclonal antibody directed 
against a distinct antigenic determinant on the intact biomarker antigen molecule and used for 
solid phase immobilization (on the microtiter wells). A secondary antibody conjugated to an 
enzyme is used as signal generator. The test sample is allowed to react sequentially with the 
two antibodies, resulting in the antigen molecules being sandwiched between the solid phase 
and enzyme-linked antibodies. 
Gold nanoparticles (AuNPs) have gained attention in the last years due to the unique 
structural, electronic, magnetic, optical and catalytic properties which have made them a very 
attractive material for biosensor systems and bioassays. The combination of biomolecules 
with AuNPs provides interesting tools for several biological components. The use of AuNPs 
for biomarker analysis is particularly explored, for example, by optical based detection 
technologies due to their extremely strong absorption and light scattering in the plasmon 
resonance wavelength regions and certain fluorescence properties. The excellent 
electroactivity of these nanomaterials allows also the use of either electrical or 
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electromechanical or electrochemical techniques as detection device, reaching low limits of 
detection (LOD). Due to their excellent biocompatibility AuNPs are also finding increasing 
application as enzyme enhancers, nanoscale building blocks and immunohistochemical 
probes. 
In this work a well established analytical format, the optical ELISA test, was combined with a 
novel concept of controlled AuNPs bioconjugation procedures aiming to improve the 
analytical performances without increasing significantly the complexity of the procedure. 
AuNPs have been adopted as multi-enzyme carriers in the enzyme-based immunoassay for 
the analysis of CA15-3 biomarker, which generated an amplified optical read-out while 
keeping low background signals. 
In addition to the standard spectrophotometric detection format, normally adopted in ELISA 
tests, an electrochemical detection has also been developed. This consisted on measuring the 
current signal generated applying a fixed potential during the reduction of the enzyme 
substrate, oxidized by the Horseradish Peroxidase (HRP) signaling enzyme in the last step of 
the ELISA. Screen-printed electrodes with silver pseudo-reference, carbon auxiliary and 
carbon working electrodes all-built-in, were used for the current measurements in a 
voltammetric cell. The current signal resulted directly proportional to the antigen 
concentration (CA 15-3) and hence it can be used as an alternative tool to quantify unknown 
samples. 
The presence of AuNPs confirmed the higher assay sensitivity due to the signal enhancement, 
already obtained with the optical detection. 
This simple concept of using AuNPs as effective signal enhancer could be easily exploited to 
improve the analytical performances of commercially available ELISA tests, especially those 
requiring high accuracy to facilitate physicians in deciding the appropriate medical treatment. 
vii 
RIASSUNTO 
La nanotecnologia è una branca della scienza applicata e della tecnologia che studia e sfrutta i 
fenomeni e i materiali esistenti solamente su scala atomica o molecolare: la scala 
nanometrica. In questi ordini di grandezza, i nanomateriali mostrano caratteristiche fisiche e 
chimiche uniche che portano a nuove proprietà a seconda delle dimensioni. Quando queste si 
riducono, infatti, il rapporto superficie/volume aumenta notevolmente, conferendo alle 
nanostrutture proprietà ottiche, meccaniche, fotochimiche, elettroniche e magnetiche non 
osservabili su scala macroscopica, il che le rende interessanti per una vasta gamma di 
applicazioni, quali ad esempio il monitoraggio ambientale, l'individuazione di agenti 
patogeni, la proteomica, la genomica, la somministrazione di farmaci, la catalisi e l‟analisi 
biologica. 
Tra i nanomatriali, le nanoparticelle hanno ricevuto grande interesse grazie alle loro proprietà 
elettroniche, ottiche, termiche, catalitiche esclusive, e alle potenziali applicazioni nel campo 
della fisica, chimica, biologia, medicina, e scienze dei materiali. Le nanoparticelle sono 
aggregati di poche centinaia fino a qualche migliaia di atomi, di dimensioni comprese tra 1 e 
100 nanometri (nm), che si comportano come unità intere in termini di trasporto e proprietà. 
Possono essere composte da un unico materiale costituente (metallo, semiconduttore o ossido) 
o da diversi materiali e venire realizzate in una varietà di forme, morfologie e fasi. 
Si definisce "biomarker" una caratteristica che è oggettivamente misurata e valutata come un 
indicatore di processi biologici fisiologici, processi patogeni, o di risposte farmacologiche a 
un intervento terapeutico. Come suggerisce la definizione, esistono diversi tipi di biomarkers 
biochimici, fisiologici, anatomici, istologici e fisici. Essi possono essere utilizzati nella pratica 
clinica per l‟identificazione del rischio o per la diagnosi di una patologia, per valutarne la 
gravità o la progressione, prevedere la prognosi, o per condurre una terapia. I markers 
tumorali sono sostanze, molto spesso proteine, che possono ritrovarsi nel sangue o nelle urine 
viii 
di un paziente affetto da cancro; esse vengono prodotte dalle stesse cellule neoplastiche, o 
dall'organismo in risposta al tumore o ad altre condizioni fisiopatologiche. 
Il punto di contatto tra nanotecnologia e biomarkers è rappresentato dalla possibilità di 
modificare e migliorare i metodi standard di identificazione e misurazione di questi ultimi, 
con l'introduzione di nanosistemi utilizzabili, ad esempio, come trasportatori di molecole, 
marcatori, mediatori di trasferimento di elettroni o catalizzatori di reazioni elettrochimiche. 
In questo lavoro l'attenzione è stata posta sull'analisi di un importante marcatore tumorale, la 
proteina CA 15-3, essenziale per monitorare le terapie del carcinoma alla mammella. 
Il tumore della mammella è il cancro più comunemente riscontrato nelle donne. Ogni anno, 
infatti, questa patologia viene diagnosticata in più di 1 milione di donne in tutto il mondo. 
CA 15-3 è una glicoproteina utilizzata principalmente per valutare i pazienti affetti da tale 
patologia. Concentrazioni plasmatiche elevate sono presenti in meno del 10% dei pazienti con 
malattia precoce e in circa il 70% dei pazienti con malattia avanzata. Valori di riferimento 
sono solitamente inferiori a 30 U/mL (unità/ml), ma a volte possono essere riscontrate 
concentrazioni pari a 100 U/mL in donne non affette da cancro. Il dosaggio del CA 15-3 viene 
comunemente effettuato per permettere ai clinici di monitorare e valutare adeguatamente la 
terapia in donne affette da neoplasie invasive, ed eventualmente suggerire la necessità di 
terapie supplementari oppure ulteriori analisi, qualora la risposta non fosse positiva. 
Questo test può essere usato anche per diagnosticare un‟eventuale recidiva della malattia, che 
può verificarsi in seguito a una terapia. 
Il dosaggio ELISA (Enzyme-Linked Immunosorbent Assay) è la tecnica più comunemente 
usata dai biologi molecolari per l‟individuazione e la quantificazione di biomarkers. I test 
ELISA in generale si basano sul principio del saggio immunoenzimatico su fase solida, con 
rilevazione spettroscopica di un reagente colorato. Tale sistema utilizza un anticorpo 
monoclonale immobilizzato su un supporto solido (i pozzetti della piastra ELISA), e diretto 
contro un determinante antigenico distinto sulla molecola dell‟antigene (biomarker). 
Un anticorpo secondario coniugato con un enzima viene invece utilizzato come generatore di 
segnale. Il campione viene lasciato reagire in maniera sequenziale con i due anticorpi, in 
questo modo le molecole di antigene rimangono intrappolate e immobilizzate tra la fase solida 
e gli anticorpi legati all‟enzima. 
Le nanoparticelle d‟oro (AuNPs) hanno assunto una certa rilevanza negli ultimi anni a causa 
delle loro particolari proprietà strutturali, elettroniche, magnetiche, ottiche e catalitiche, che le 
hanno rese un materiale molto interessante per i sistemi di biosensori e i metodi di analisi 
ix 
biologica. La combinazione di biomolecole con AuNPs offre strumenti interessanti per 
diverse componenti biologiche. L'uso di AuNPs per l‟analisi di biomarkers è particolarmente 
sfruttato, per esempio, dalle tecnologie basate su rilevamento ottico, a causa della loro 
capacità di assorbimento e dispersione della luce estremamente forte e delle proprietà di 
fluorescenza. L‟eccellente elettroattività di questi nanomateriali permette anche l'utilizzo di 
tecniche sia elettriche, sia elettromeccaniche, sia elettrochimiche come metodi di rilevazione, 
raggiungendo limiti di rilevabilità (LOD) molto bassi. Grazie alla loro eccellente 
biocompatibilità, le AuNPs stanno trovando impiego sempre più frequentemente anche come 
amplificatori del segnale enzimatico, come materiali di partenza nanometrici e come sonde 
immunoistochimiche. 
In questo progetto di tesi, un modello analitico ben definito, il test ELISA, è stato combinato e 
integrato con una procedura di coniugazione di biomolecole con nanoparticelle d‟oro, con 
l‟obbiettivo di migliorare le prestazioni analitiche senza aumentare in modo significativo la 
complessità della procedura. 
Le AuNPs sono state usate come trasportatori multi-enzima nel dosaggio ELISA per l'analisi 
del biomarker CA15-3, che ha generato un segnale ottico amplificato, mantenendo “segnali di 
fondo” trascurabili. 
Oltre al formato standard di rilevazione spettrofotometrica, normalmente utilizzata nei test 
ELISA, è stato sviluppato un metodo di rilevazione elettrochimica. Questo consiste nel 
misurare il segnale di corrente generato applicando un potenziale fisso durante la riduzione 
del substrato dell'enzima, ossidato dall‟enzima perossidasi di rafano (HRP) nell‟ultimo 
passaggio dell‟ELISA. Per misurare il flusso di corrente all‟interno della cella voltammetrica 
sono stati utilizzati elettrodi serigrafati, composti da un elettrodo di riferimento in argento, un 
elettrodo di lavoro in carbonio e un elettrodo ausiliario anch‟esso in carbonio. 
Il segnale di corrente è risultato direttamente proporzionale alla concentrazione dell‟antigene 
(CA 15-3) e, quindi, può essere usato come espediente alternativo per la quantificazione di 
campioni incogniti. 
La presenza delle AuNPs ha confermato una maggiore sensibilità del saggio, dovuta 
all‟amplificazione del segnale, già ottenuta con la rilevazione ottica. 
Questo semplice utilizzo di AuNPs in qualità di amplificatori efficaci del segnale, potrebbe 
essere facilmente sfruttato per migliorare le prestazioni analitiche dei test ELISA disponibili 
in commercio, in particolare quelli che richiedono elevata precisione per agevolare i clinici 
nella scelta della terapia adeguata. 
Chapter 1 - GENERAL INTRODUCTION 1 
Chapter 1 - GENERAL INTRODUCTION 
1.1 Clinical analysis and biomarkers 
1.1.1 Clinical analysis: an overview 
The purpose of clinical laboratories is the provision of accurate and precise test results for 
patient care in a short and acceptable time. In general terms laboratory tests can be divided 
into three large classes depending upon the clinical use of the data: a) screening tests, b) 
monitoring tests, c) diagnostic tests. 
A) Screening tests aim at separating normal from abnormal subjects. Characteristically they 
are semiquantitative or quantitative and should have low false-negative rates. In other words 
they are designed not to miss abnormal values. For the most part they are nondiagnostic and if 
possible require additional confirmatory studies. Typical examples of such screening tests are 
the automated blood count (without a blood film examination), a dipstick urinalysis, or a stool 
test for occult blood. If a screening test is positive, the diagnostic test can confirm or exclude 
the presence of a disease or condition. 
B) Monitoring tests are quantitative and must be very precise in order to accomplish their 
goals. Blood glucose testing in diabetics is probably the most widely employed monitoring 
procedure. Errors can lead to the mistaken injection of insulin or conversely to the infusion of 
excess glucose in the diabetic patient. Another common example of a laboratory monitoring 
procedure is the prothrombin time when used to monitor oral anticoagulation. 
Therapeutic drug monitoring is yet another field which has grown rapidly in the last few 
years. Again, errors in testing may lead to significant clinical problems. 
C) Diagnostic tests can be either qualitative or quantitative but above all they must have very 
low false-positive rates. An example of a qualitative diagnostic test is a microbiologic culture 
and sensitivity testing of the cultured organism. Diagnostic immunology tests are also 
frequently qualitative in nature. Think, for example, of immunologic tests for syphilis or 
human immunodeficiency virus (HIV). There are many quantitative tests. Examples include 
Chapter 1 - GENERAL INTRODUCTION 2 
low granulocyte counts which define neutropenia, low platelet counts defining 
thrombocytopenia, and low or high erythrocyte counts leading to diagnoses of anemia or 
polycythemia. Errors in such tests can lead to significant clinical misadventures by either 
1
withholding or providing inappropriate therapy. 
1.1.2 The role of biomarkers in clinical analysis 
Biomarker is a much broader term, including everything from binding affinity, to genotype, to 
measures that predict clinical outcome. A National Institutes of Health biomarker working 
group has defined a biomarker as “a characteristic that is objectively measured and evaluated 
as an indicator of normal biologic processes, pathogenic processes, or pharmacologic 
responses to a therapeutic intervention”. Body temperature, for example, is a well-known 
biomarker for fever, and blood pressure is considered an effective biomarker for risk of 
stroke. Cholesterol is accepted as a biomarker of cardiovascular risk. As the definition 
suggests, there are many types of biomarkers, including biochemical, physiologic, anatomic, 
2,3,4
histologic and physical markers. Although the term “biomarker” is relatively new, 
biomarkers have been used in pre-clinical research and clinical diagnosis for a considerable 
time. In fact they can be used in clinical practice to identify risk for or diagnose a disease, 
separate the more serious patients from the less serious ones, assess disease severity or 
progression, predict prognosis, or guide treatment. In drug development biomarkers may be 
used to help determine how a drug works in the body, to determine a biologically effective 
dose of a drug, to help assess whether a drug is safe or effective, and to help identify patients 
most likely to respond to a treatment, or are least likely to suffer an adverse event when 
treated with a drug. Biomarkers can sometimes be used as part of the approval process for a 
4,5
drug or treatment, to help make a decision. 
1.1.3 Tumor markers 
Within the last decade a large number of laboratory tests have become available which 
collectively have been labeled "tumor markers." These studies have been used for screening 
1
as well as diagnostic and monitoring purpose. 
Chapter 1 - GENERAL INTRODUCTION 3 
1.1.3.1 Definition of tumor marker 
Tumor marker is a substance that can be found in the body when cancer is present or in 
progress. It is usually found in the blood or urine and it can be a product of the cancer cells 
6
themselves or of the body in response to cancer or other conditions. There are many different 
tumor markers: most of them are generic proteins, but they can be also hormones, enzymes, 
metabolites, immunoglobulins, tumor associated antigens, oncogenes and other substances 
that are helpful in the detection and diagnosis of certain cancers; some of them are seen only 
in a single type of cancer, while others can be found in many types of cancer. Sometimes non-
cancerous diseases can also cause levels of certain tumor markers to be higher than normal, 
and not every person with cancer may have higher levels of a tumor marker. For these 
reasons, currently no tumor marker has characteristics such as sensitivity and specificity to be 
used by itself in early diagnosis and/or mass screening of cancer. Instead, in combination with 
other instrumental investigations, they can help in the differential diagnosis between benign 
6,7,8,9
and malignant and in the identification of relapse. 
1.1.3.2 Classification of tumor markers 
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There are four main groups of tumor markers: 
1) Structural molecules: Structural molecules are commonly found on the cell surface. Hence, 
they are of little value in identifying tumor type. Examples of tumor makers which are 
structural molecules are carcinoembryonic antigen (CEA), mucins like CA 19-9, CA 15-3 and 
CA125, β-2-microglobulin and cytokeratins like CYFRA 21-1 and tissue polypeptide antigen. 
2) Secretion products and enzymes: Examples of secretion products and enzymes include α-
foetoprotein (AFP), human chorionic gonadotrophin (HCG), paraproteins and Bence-Jones 
protein, prostate-specific antigen (PSA), neuron-specific enolase (NSE) and placental-like 
alkaline phosphatase. 
3) Non-specific markers of cell turnover: These include neopterin and thymidine kinase. 
4) Cellular markers: Examples of cellular markers are Philadelphia chromosome, oncogenes, 
tumor suppressor genes, oestrogen receptor, progesterone receptor, epidermal growth factor 
receptor and c-erbB-2.