Dissertations / Theses on the topic 'Biomarkers detection'

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2010.<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references (p. 131-147).<br>Prostate cancer persists as a major public health issue in the United States and remains the second leading cause of cancer death in men. Early detection and disease monitoring in prostate cancer can significantly improve a patient's prognosis. The advent of prostate-specific antigen (PSA) screening has allowed physicians to monitor the levels of a specific protein, or biomarker, as a correlate of disease progression. This thesis focuses on optical detection of prostate tumors through the development of biomarker-targeted molecular imaging probes. In the first part of this work, engineered human prostate cancer cell lines were developed and characterized to determine the dynamics of post-translational processing for PSA proteolytic activity and to establish potential small animal models for validating protease-activatable imaging probes. Target-activatable gold nanoparticle imaging probes that can be self-assembled in a one-step reaction were then developed to detect biomarker proteases in vivo. The activated probes demonstrated a 5 to 8-fold fluorescence signal amplification, extended circulation time, and high image contrast in a mouse tumor model. Lastly, differential phage display selection was performed on human prostate cancer cells with low and high metastatic potentials to (1) identify cell-surface biomarkers specific to highly aggressive tumors, and (2) develop molecular imaging probes for detecting prostate cancer metastases.<br>(cont.) One peptide, LN4P-1, demonstrated preferential binding to highly metastatic PC3M-LN4 cells and identified a highly expressed protein on their cell surface. Fluorescently labeled LN4P-1 was able to detect PC3MLN4 tumors in vivo. In summary, this thesis outlines the development of molecular imaging probes for targeting tumors both at the primary site, through evaluation of biomarker protease activity, and at the metastatic site, through affinity-based analysis of biomarker expression.<br>by Chunyao Jenny Mu.<br>Ph.D.

In recent years the rising cost and increased regulation within the U.S. healthcare system have caused medical laboratory tests to become more costly and more frequently required. As a result, insurance premiums are rising, and small independent laboratories are threatened with closure as their already narrow margins dwindle. Concurrently, there have been several incidents of contaminants and impurities in pharmaceutical drugs causing hundreds of deaths and thousands of illnesses. These challenges substantiate the need for simple and cost-effective screening tests for the presence of disease biomarkers, as well as for contaminants and impurities present in pharmaceutical drugs. The following disquisition reports three independent studies, each with the development of simple screening tools as its objective. Paper 1 reports the use of fluorescent lipid nanoparticles (liposomes) to detect changes in the species and concentrations of glycosaminoglycans (GAGs) in solution. We conclude that the emission intensity from the present fluorophores changes in response to increasing concentrations of GAGs, and can distinguish between serum from a healthy patient and serum having the same GAG concentrations as an Alzheimer's disease patient (simulated). Paper 2 reports the use of lipid nanoparticles to detect dangerous over-sulfated contaminants in pharmaceutical heparin. We report that liposomes in the presence of heparin or over-sulfated contaminants and Mg2+ ions form aggregates, and the size and zeta potential of these aggregates is dependent on the heparin/contaminant present. Further, the variation in aggregate zeta potential varies significantly upon heparin contamination, and may be used to detect 0.5% contaminant by weight. Paper 3 reports a clinical study to validate the presence of ADAM 12 (a disintegrin and metalloproteinase) enzyme in urine as a biomarker for breast cancer detection and diagnosis, as well as to monitor the effects of tumor removal on the urinary levels of this enzyme. We find no significant differences between recently diagnosed cancer patients (having undergone no treatment for cancer) and age-matched controls having no cancer present. Significant increases in urinary ADAM 12 only occur following surgery. Overall, we conclude that it is unlikely that a screen for urinary ADAM 12 will be useful for the diagnosis of breast cancer.<br>National Institutes of Health (NIH)<br>North Dakota State University. Pharmaceutical Sciences<br>Sanford Health<br>American Foundation for Pharmaceutical Education

Las enfermedades infecciosas suponen una gran amenaza para la salud mundial debido a la rápida diseminación y adaptación de los patógenos. El papel principal del diagnóstico clínico es identificar de forma fehaciente una enfermedad en un paciente. Los dispositivos de diagnóstico rápido permiten detectar enfermedades y monitorearlas de forma confiable en cualquier centro sanitario. Entre estos dispositivos, los biosensores electroquímicos presentan una alta sensibilidad y especificidad así como una instrumentación sencilla, pudiendo expandirse fácilmente a plataformas de detección múltiple. Además, la integración de partículas magnéticas (MPs) en métodos de diagnóstico rápido incrementa la sensibilidad y especificidad debido su capacidad para aislar y preconcentrar una molécula diana cuando éstas son modificadas con elementos de bioreconocimiento específico. Así, las MPs modificadas pueden unirse específicamente a biomarcadores y concentrarlos de muestras complejas bajo la actuación magnética, eliminando posibles interferencias. En esta tesis se presenta el desarrollo de estrategias de diagnóstico basados en tecnologías emergentes, asequibles y que requieren un entrenamiento mínimo de los usuarios finales, tales como los biosensores electroquímicos con accionamiento magnético. Primero, se presentan dos tipos de pruebas diagnósticas para la cuantificación de linfocitos CD4 en sangre entera, usando MPs, para el seguimiento rápido de pacientes con HIV en entornos de bajos recursos. Se describen dos formatos, uno en formato tipo ELISA con detección óptica y otro usando electrodos de grafito-epoxi con biosensado electroquímico. En ambos casos la estrategia involucra a) el aislamiento de células CD4 usando MPs-antiCD3 y su marcación utilizando anticuerpos antiCD4 biotinilados; b) la marcación enzimática con estreptavidina-peroxidasa; y c) la detección basada en la actividad enzimática. Esta doble marcación (a través de los receptores CD3 yCD4) no sólo evita interferencias de otras células que expresan alguno de estos receptores, sino que aumenta la especificidad del ensayo. Segundo, se describe un ensayo de liberación de interferon-basado en la detección electroquímica de dicho trascripto producido por linfocitos T previamente aislados de sangre. Este test utiliza MPs para el aislamiento y preconcentración de tres dianas diferentes (linfocitos T, mRNA y ADN doblemente marcado) en el mismo ensayo. Primero, los linfocitos T se aislan usando MPs-antiCD3. En segundo lugar, el mRNA de los linfocitos se preconcentra sobre MPs modificadas con polidT mediante unión a la cola de poli(A). Posteriormente se retrotranscribe el mRNA y se amplifica el cDNA mediante PCR múltiplex con marcación doble para la amplificación de IFN- y GAPDH. Finalmente, se inmovilizan los amplicones biotinilados en MPs-estreptavidina, y se realiza el genosensado electroquímico para la detección de IFN- a través del otro marcador del cebador. Esta estrategia se propone como alternativa a los ensayos de liberación de IFN- que se usan en la actualidad para la identificación de la Tuberculosis. Por último, se presenta el diseño de un test de diagnóstico rápido, específico y altamente sensible basado en la amplificación isotérmica sobre MPs con detección electroquímica. Las técnicas de amplificación isotérmicas han surgido como una alternativa a la PCR para la identificación de microorganismos infecciosos, debido a la barrera que éstos últimos muestran para su implementación en entornos de bajos recursos. El último capítulo presenta la detección electroquímica de ADN usando sondas candado con amplificación isotérmica de círculo rodante y amplificación círculo a círculo. Esta estrategia ha demostrado ser una poderosa herramienta para la detección específica y sensible de ácidos nucleicos para su aplicación en el diagnóstico clínico. Los biosensores desarrollados en esta tesis representan una gran promesa para la detección de forma más rápida, simple y económica comparado con los métodos tradicionales de diagnóstico de enfermedades infecciosas. Además, las estrategias desarrolladas en esta tesis demuestran un gran potencial para su aplicación en entornos de bajos recursos.<br>Infectious diseases are becoming a major threat worldwide due to the fast dissemination and adaptation of pathogens favored by the unrestricted globalization. The primary role of diagnostics is to identify a disease. The rapid identification of a disease allows the patient to be placed on a specific antimicrobial therapy and avoid prolonged management on empiric, potentially inappropriate drug. Therefore, point-of-care (POC) devices that can reliably detect and/or monitor diseases would result in an improved care, and minimization of patient and societal cost of illness. Among them, electrochemical biosensors have the advantage of high sensitivity/specificity as well as simplicity of instrumentation, and can be easily expanded to multiplex detection platform. Furthermore, the integration of magnetic particles (MPs) in POC tests provides an even increased sensitivity and specificity due to the isolation and preconcentration of the target, whether MPs are modified with a specific recognition biomolecule. Modified-MPs can thus specifically bind the biomarkers and preconcentrate them from the complex specimen under magnetic actuation, preventing interferents before testing. Affordable emerging technologies requiring minimal training for final users, such as magnetic actuated electrochemical biosensors, are presented in this dissertation. Firstly, two simple diagnostic tests for CD4+ T lymphocytes quantification, directly in whole blood, and based on magnetic particles are presented. The assay is performed in an ELISA-like format for the optical detection or using graphite-epoxy electrodes for the electrochemical biosensing strategy. In both cases, the strategy has involved three main steps: a) immunomagnetic separation of CD4+ cells by antiCD3-MPs and labeling by using biotinylated antiCD4 antibody; b) enzymatic labeling; and c) detection based on the peroxidase activity. The dual labeling (CD3 and CD4 receptor) not only avoids interferences of other cells, but also increases the specificity of the assay. Thus, the development and evaluation of magnetic-actuated rapid HIV diagnostic platforms appropriate for their use in low resource settings for the following-up of patients under treatment is demonstrated. Secondly, an interferon- release assay based on electrochemical detection for interferon- transcript detection produced by isolated T lymphocytes is described. This approach also involves the integration of MPs for the isolation and preconcentration of three different targets (including whole T lymphocytes, mRNA transcripts and double-tagged DNA) in the same test. Accordingly, T lymphocytes are isolated from whole blood using antiCD3-MPs. Secondly, mRNA presenting poly(A) tail is preconcentrated on polydT-MPs from T lymphocyte. Afterward, mRNA is retrotranscripted and cDNA amplified by multiplex double-tagging PCR for the specific amplification of IFN- and GAPDH. Finally, one of the tags of the primers is used for the amplicons immobilization on streptavidin-MPs as support, while the electrochemical magneto-genosensing for transcript detection is performed using the other tag. This strategy results in an alternative for IFN- release assays, which can be used for identifying infectious states such as Tuberculosis. Finally, the design of a diagnostic test involving a rapid, specific and highly sensitive procedure based on isothermal amplification on MPs with electrochemical readout is presented. Isothermal amplification techniques are emerging as good candidates to replace PCR for the identification of infectious microorganism, since PCR-based method can be a critical barrier in low resource settings. An electrochemical DNA detection using padlock probes and the subsequent amplification with rolling circle and circle to circle amplification is presented in Chapter 6. This strategy has demonstrated to be a powerful combination for highly specific and sensitive nucleic acid detection that can be applied in clinical diagnosis. The electrochemical biosensors developed in this dissertation, offers considerable promise for obtaining information in a faster, simpler and cheaper manner compared to traditional methods for infectious disease diagnosis. Moreover, the strategies possess great potential in many applications, in low resource settings.

The introduction of prostate-specific antigen (PSA) testing about 3 decades ago led to the possibility of early detection of prostate cancer (PCa). Although PSA testing reduced the mortality rate, it is also associated with high risk of over diagnosis in patients with and without PCa. Despite the current drawbacks, it would be a challenge to replace PSA testing entirely. Instead, there is a need to develop parallel testing of other potential biomarkers that can complement the results from PSA tests. To address alternative biomarker sensing, this thesis highlights on the development of oligonucleotide-based biosensors for the detection of different biomarkers of PCa. Using PSA as a gold standard, the first study of this dissertation investigates the use of DNA aptamers to detect PSA using electrochemical impedance spectroscopy (EIS). The study compares 6-mercapto 1-hexanol chemistry with sulfo-betaine chemistry for the development of PSA aptasensor in terms of performance and selectivity. The second study focuses on glycoprofiling in order to complement PSA quantification as an additional information for reliable PCa diagnosis. This strategy was developed in a microfluidic channel with an optical read out using chemiluminescence. This study addresses one of the major problems of cross-reactivity with lectins in glycoprofiling, which can be solved using DNA aptamers. A third study concentrates on the development of an aptasensor for Alpha-Methylacyl-CoA Racemase (AMACR). AMACR has been reported for its high specificity and sensitivity to PCa. For the fabrication of the biosensor, a new strategy using polyethylene glycol was developed by electrochemical grafting it to a polypyrrole film. Since PCa diagnosis can be improved by looking at different biomarkers, an electrochemical platform for miRNA/DNA detection using a gold nanoparticle amplification strategy was also investigated. The sensor was fabricated using peptide nucleic acids (PNA) probes on gold electrodes. The study presents non-Faradaic EIS and amperometric techniques in order to exploit the inherent charges of nucleic acids. In conclusion, this thesis wants to serve as a potential orientation for overcoming the shortcomings of the current PCa testing and contribute towards the development of oligonucleotide-based biosensors for PCa biomarker detection and hopefully enhance the diagnosis and prognosis of PCa.

Magister Scientiae - MSc (Chemistry)<br>Pulmonary Tuberculosis (TB) a disease second to HIV/AIDS is a global health problem that arises in two states; as an active state and as a latent state. Diagnosis of active TB is tedious and requires expensive procedures since there is no recognizable method for the sole detection of active TB. The current diagnosis consists of chest X-rays and multiple sputum cultures used for acid-fast bacilli detection. The TB diagnosis of children is particularly difficult which further complicates the diagnosis. Thus, rapid identification of this pathogen is important for the treatment and control of this infection to allow effective and timely therapy. In an effort to solve this issue, this study reports the development of immunosensors constructed with electroactive layers of amino groups functionalized graphene oxide (GO) doped respectively with green synthesized zinc oxide (ZnO NPs) nanoparticles and silver (Ag NPs) nanoparticles on glassy carbon electrodes. The surface morphology of GO, ZnO NPs, Ag NPs and their composites was revealed by employing High-Resolution Transmission Electron Microscopy (HR-TEM) and High-Resolution Scanning Electron Microscopy (HR-SEM) while the composition and structure of these materials were studied using Fourier Transform Infra-Red Spectroscopy (FTIR). The resultant graphene oxide-metallic composites were covalently attached with CFP-10 and/or ESAT-6 antibodies to achieve the electrochemical detection. The immunosensor was then used for the impedimetric and amperometric detection of anti-CFP-10 and/or anti-ESAT-6 antigens in standard solutions.

Neurological diseases such as Alzheimer's, Parkinson's, MS, which are common around the world and particularly in developed countries with high proportion of elderly, stimulate the development of bio-sensors for early diagnostics of such diseases. For instance the treatment of Alzheimer's patients constitutes a substantial proportion of NHS budget nowadays. Therefore, the development of highly sensitive optical devices for early diagnoses of Alzheimer's disease (AD) will be beneficial to society. This PhD is mainly dedicated to the application of the method of Spectroscopic Ellipsometry for AD diagnostics. Total Internal Reflection Ellipsometry (TIRE) was utilized in this work as an immunosensor for detection of Amyloid Precursor Protein 770 (APP[770]) and beta amyloid peptide (Abeta[1-16)]. The detection of low concentrations of APP[770] in a complex medium containing other proteins, salts, and amino acids was achieved using the method of TIRE in direct immuno assay with monoclonal DE2 antibodies. The calibration of TIRE with a complementary QCM measurements in air allowed the evaluation of (originally unknown) concentration of APP[770] as 121 pmol/l. The immune reaction between APP[770] and DE2 antibodies was also tested using QCM technique operating in liquid. The application of TIRE was extended to the detection of much smaller peptide Abeta[1-16] which bind to the same DE2 antibody. The results were very encouraging since low concentrations (0.05ng/ml) of Abeta[1-6] were detected; this showed good prospects for detection of Abeta[40-42], an actual marker of AD.The second part of this work was dedicated to detection of mycotoxins, a hazardous contaminant in agriculture products (grains) and associated food and feed. Two mycotoxins, namely Aflatoxins B1 and Zearalenone, were detected in TIRE direct immunoassay. Zearalenone was also detected using TIRE competitive assay. The obtained limits of detection of 0.04 ng/ml for Aflatoxin and 0.1 ng/ml for Zearalenone are well below the legislation limit. Such remarkable results are due to a combination of high sensitivity of TIRE method and the aggregation of hydrophobic molecules of mycotoxins in aqueous solutions. A new method of purification of substances contaminated with mycotoxins based on the use of polyelectrolyte microcapsules functionalized with specific antibodies was successfully tested in this work.

Cancer biomarkers may hold the key for the early detection of cancer, distinguishing between benign and malignant cells, and differentiating tumor types. The detection of cancer biomarkers can be used for cancer diagnosis, monitoring the response to therapy, and providing real-time prognostic information for cancer patients. Most of the traditional cancer biomarker detections are based on specific antibodies or expensive instrumentations/complex operations. In this dissertation, we have developed aptamer-based bioassays for visual detection of cancer protein biomarkers with low-cost and short assay time. Aptamers with specific sequences are immobilized on gold nanoparticle (AuNP) surface through self-assembling process. Combining the excellent molecular recognition properties of aptamers and the unique optical properties of AuNPs, colorimetric assay for carcinoembryonic antigen (CEA) and mucin 1 (MUC1) (breast cancer biomarkers) and lateral flow assay for platelet-derived growth factor (PDGF) and thrombin have been developed. The methods were applied to detect protein biomarkers in human plasma and blood successfully. The sensitivities of the assays were further improved by using enzyme-coated AuNP dual labels and designing the cross-flow test strips. The developed approaches have the potential to be extended for detecting other biomarkers, and show great promise for point-of-care or in-field detection.<br>National Science Foundation (NSF)<br>NDEPSCoR

El càncer de pròstata (CP), és la segona causa de mort per malaltia oncològica en els homes del món occidental. S’estima que un de cada sis homes desenvoluparà un càncer d’aquest tipus al llarg de la seva vida. El CP afecta com el seu nom indica, a la pròstata. La pròstata, és un òrgan glandular de l’aparell genital-urinari masculí. Té la mida d’una nou i es localitza sota de la bufeta, envoltant la uretra i davant del recte. La seva funció és secretar productes que s’afegiran al líquid seminal amb la finalitat de nodrir i protegir els espermatozous. El actual diagnòstic del CP es basa en una triada diagnòstica que consta de; l’anàlisi dels nivells de PSA (Prostate Specific Antigen) en sèrum, el tacte rectal (TR) i finalment la biòpsia prostàtica. Quan els nivells de PSA en sèrum es situen per sobre de 4 ng/mL i/o el TR és sospitós, l’uròleg pot estimar quina és la probabilitat que el pacient estigui afectat per un CP i per tant decidir la necessitat de practicar o no una biòpsia prostàtica, que permetrà establir el diagnòstic definitiu. La introducció del test del PSA, a finals dels anys 80, s'ha traduït en una millora del diagnòstic precoç del CP, moment en el qual les opcions de tractament per el són eficaces. No obstant això, malgrat aquesta detecció primerenca, la mortalitat per CP no ha disminuït significativament en els últims anys. El principal problema del PSA com a marcador d’screening és que aquest presenta un nivell d’especificitat baix (30% aprox) i alhora, un valor predictiu negatiu baix. Això es tradueix en que al voltant d'un terç de tots els homes sotmesos a una biòpsia seran positius per CP. Com a resultat dels seus persistents nivells sèrics de PSA, però els resultats negatius de la biòpsia, aquests homes es sotmeten a repetides biòpsies. Tot i el importants avenços en la investigació de biomarcadors de CP, alguns homes encara estan sobre-diagnosticats de CP “indolent”, mentre que d’altres moren de malaltia agressiva que s'ha diagnosticat massa tard. Aquesta situació es coneix com "dilema diagnòstic". És per tot això, que el càncer de pròstata, es beneficiaria de l’existència de nous marcadors d’screening més específics i alhora d’un diagnòstic menys invasiu. Per altra banda, una millora en el diagnòstic evitaria un gran nombre de biòpsies innecessàries i conseqüentment un important estalvi econòmic en el cost sanitari actual. La recerca de nous marcadors en el càncer de pròstata suposa un camp de treball important en la detecció precoç d’aquest tipus de càncer. Donada la situació de la pròstata a l’organisme, sota la bufeta i envoltant la uretra, les secrecions i inclús les mateixes cèl·lules prostàtiques, ja siguin normals o malignes, poden trobar-se presents en l’orina. És per això que considerem l’orina com una font important d’informació, a través de la qual es podria arribar a determinar quina situació s’està donant a l’òrgan en qüestió. Altres estudis evidencien l’existència de potencials biomarcadors en l’orina que podrien ajudar en la millora del diagnòstic del CP. A nosaltres ens ocupa l’estudi d’aquelles molècules de RNA així com de les molècules protèiques que es troben a l’orina. Suposem doncs, que un massatge prostàtic enriqueix la mostra d’orina de tot tipus de molècules proteiques. Així doncs, la nostra hipòtesi de treball recolza que, l’orina després d’un massatge prostàtic pot ser el fluid ideal per a la recerca de nous biomarcadors capaços de discriminar entre pacients amb o sense càncer de pròstata. L’objectiu principal de l’estudi és diagnosticar el CP assimptomàtic per us sistema minimament invasiu, que consisteix en l’anàlisi de l'ARN o de la proteïna en l'orina obtinguda després d’un massatge prostàtic i, superar la baixa especificitat de l’actual sistema d’screening (PSA en sèrum) mitjançant l'ús de biomarcadors addicionals (RNA o proteina) per tal de reduir el nombre de biòpsies innecessàries (reduir els costos socio-econòmics, així com la reducció dels efectes secundaris no desitjats). 1. ANÀLISI TRANSCRIPTÒMICA: 1a) “PSGR and PCA3 as Biomarkers for the Detection of Prostate Cancer in Urine” Rigau M et al., Prostate. 2010 Dec 1;70(16):1760-7. En el primer estudi, mitjançant l’anàlisi de transcripts per RTqPCR en 215 (34% amb CP) mostres de sediements d’orines de pacients (obtingudes després d’un massatge prostàtic), es va determinar la utilitat de PSGR (Prostate Specific G-coupled receptor), previament descrit com a sobre-expressat en mostres de teixit de CP, com un nou biomarcador, comparable a PCA3 (ja conegut), per a la detecció del CP en orines (taula 1). Per l'anàlisi de la corba ROC vàrem determinar els valors de l’Area sota la corba (AUC) de PSGR (AUC 0,68) i PCA3 (AUC 0,66). Fixant la sensibilitat a 95%, aconseguim un valor d’especificitat de 15% per PSGR i el 17% de PCA3. Alhora vàrem veure que al combinar els dos biomarcadors (PSGRvPCA3) mitjançant un anàlisi “multiROC”, els resultats van millorar significativament en termes de rendiment del biomarcador (AUC 0,73) com de la seva especificitat (34%). En conclusió, podem dir que PSGR presenta un comportament similar al biomarcador estàndard per CP en orina (PCA3), però, és necessàri la realització d’estudis futurs per millorar encara més el rendiment d'aquesta prova. 1b) “A Three-Gene panel on urine increases PSA specificity in the detection of prostate cancer” Rigau et al., Prostate. 2011 Apr 25. doi: 10.1002/pros.21390. En un segon estudi, vàrem estudiar la possibilitat de multiplexar diferents biomarcadors, per tal de ser capaços de guanyat especifcitat sense perdre sensibilitat en la detecció del CP. Donada l’heterogeneïtat del CP la idea d’utilitzar diferents marcadors per a la seva detecció es presenta atractiva. Mitjançant l’anàlisi de transcripts, per RTqPCR, en 215 (37% amb CP) mostres de sediements d’orines de pacients (obtingudes després d’un massatge prostàtic), vàrem determinar la utilitat de PSMA (Prostate Specific Membrane Antigen), un marcador descrit anteriorment com a sobre-expressat en el CP, com a biomarcadors per a la detecció de CP en orina. L’anàlisi de corbes ROC revela un rendiment similar PSMA (AUC 0,62), PSGR (AUC 0,65) i PCA3 (AUC 0,60) (Taula 1). Per tal de millorar l'eficàcia diagnòstica d'aquests biomarcadors es van combinar PSMAvPSGRvPCA3 (3M), utilitzant un anàlisi de multiROC. L’AUC del 3M (AUCm) (0,74) millora notablement, en comparació amb els valors individuals per a cada un dels marcadors. D'altra banda, si combinem 3M amb el valor de PSA en sang l’AUCm (0,77) va ser encara millor (taula 11). Si fixem la sensibilitat a 96%, l'especificitat del model 3M manté una especificitat del 34%, mentre que no es va observar augment en d’aquest valor quan ho combinem amb PSA en sang (34%) (Taula 11). El comportament d'aquests tres marcadors en una subpoblació de l’estudi que presentava nivells de PSA en sang entre 4-10 ng/mL i sense informació d’una biòpsia prèvia "zona gris del PSA" és de PSMA (AUC 0,74), PSGR (AUC 0,66) i PCA3 (AUC 0,61). Per al model combinat (3M), l’AUCm és de 0,82 (Taula 11). Fixant la sensibilitat al 96%, l'especificitat del model combinat (3M) manté una especificitat del 50%. En conclusió, aquests resultats demostren que la combiació de diferents marcadors proporciona una major especificitat a la prova sense comprometre el valor de sensibilitat. Expresat en el nombre de biopsies que s’haurien pogut estalviar, els valors se situen al voltant del 34% de biòpsies estalviades. 1c) “Behavior of PCA3 gene in the urine of men with high grade prostatic intraepithelial neoplasia” Morote and Rigau et al., World J Urol. 2010 Dec;28(6):677-80. El tercer estudi, està centrat en la utilitat, del ja conegut biomarcador per CP en orina (PCA3), per a l’identificació una lessió pre-maligna a la pròstata, així com la seva utilitat en la selecció dels homes amb biòpsies negatives, que requereixen la repetició del procediment. La troballa de les lesions de HGPIN (High grade preneoplastic intraepithelial lesion) és una indicació freqüent de la repetició de la biòpsia. Tot i la controvèrsia al voltant de la definició de HGPIN com a lessió pre-maligna i la necessitat de repetició de la biòpsia, encara avui en el nostre hospital s’indica el control exhaustiu del pacient, així com la repetició de la biòpsia en un periode de relativament curt de temps. Per a aquest estudi es van seleccionar un subgrup de pacients amb HGPIN (n=64) i com a grups control, es van seleccionar els homes amb CP (n=83) i homes amb patologia benigne de la pròstata (BP) (n=97). Després de l’anàlisi dels transcripts de PCA3 per RTqPCR en mostres de sediements d’orines de pacients (obtingudes després d’un massatge prostàtic), i de l'anàlisi de la corba ROC, es van obtenir els següents resultats; HGPIN vs CP (AUC 0,63) i BP vs PC (AUC 0,71). Aquests resultats demostren la capacitat dels transcripts de PCA3, en orina obtinguda deprés d’un massatge prostàtic, per a l’identificació d’homes amb CP així com la identificació de pacients amb HGPIN. 2. ANÀLISI PROTEÒMICA COMPARATIVA L’objectiu principal d’aquesta segona part és determinar un perfil proteòmic a l’orina capaç de diferenciar entre pacients amb CP i controls sans. Per dur a terme aquest objectiu ens vàrem plantejar fer una part inicial de discovery, que vàrem complementar amb “data mining” i finalment vàrem verificar mitjançant una tècnica proteòmica independent. 2a) Per la part de discovery es va dur a terme l’anàlisi comparativa de “Differential in Gel Electrophoresis (DIGE). Es van fer dos experiments independents, amb mostres de sobrenedants d’orines (on trobem la fracció soluble de les proteïnes). En el primer experiment es van comparar 6 mostres de proteïna total de pacients amb CP i 6 mostres controls. En el segon experiment es van comparar 9 mostres, de proteïna pre-tractada amb una tècnica de depleció per les proteïnes majoritàries, de pacients amb CP i 9 mostres de pacients control. Un total de 24 proteïnes (9 sobre- i 15 infra-expresades) vàren ser identificades per MALDI-TOF. Una anàlisi informàtic de les proteïnes va mostrar que la majoria d'aquestes proteïnes identificades es secreten els components de diverses xarxes ben conegudes, el càncer funcionals i la inflamació, com ara NFКB, PDGFBβ i β-catenina. D'altra banda, a causa del fet que hem utilitzat sobrenadants d'orina, on podem trobar proteïnes solubles secretades, la majoria de les proteïnes identificades (62%) es van localitzar en l'espai extracelular. Mitjançant una tècnica de proteòmica dirigida (Selected Reaction Monitoring: SRM) es van quantificar de forma relativa, sense l’adició d’estandards interns marcats, un total de 15 (dels 24 biomarcadors) en una població independent de 50 mostres (38% amb CP). Després d'una anàlisi de regressió logística de les dades obtingudes a través de l'assaig de SRM, es va obtenir un panell de set pèptids que corresponen a 5 proteïnes, capaços de distingir entre les mostres de CP i mostres controls amb una sensibilitat del 95% i una especificitat del 78 %. 2b) La segona part de l’estudi de proteómicas es basa en la verificació de les proteïnes prèviament qualificades i alhora l’estandarització dels mètodes de preparació de mostres per al seu posterior anàlisis mitjançant la tècnica de SRM. Les 5 proteïnes que formen part del panell identificat, juntament amb altres proteïnes biologicament rellevants, definides a la nostre fase de discovery, i juntament amb altres proteïnes descrites a la literatura, vàren ser escollides per una segona fase de qualificació i verificació en una població independent de 107 pacients, mitjançant un assaig de SRM en el qual es pretén quantificar de forma absoluta (adició de pèptids estàndards marcats), un total de 42 proteïnes. Prèviament a l’evaluació d’aquestes proteïnes, es va dur a terme una estandarització del protocol d’extracció de proteïna en mostres d’orina, amb l’objectiu de maximitzar la senyal obtinguda en els experiments de SRM. La precipitació amb Acetonitril (a temperatura ambient) seguida de la digestió de les proteïnes en solució amb condicions de ratio 1:10 (trypsine:protein), “over-night” i l’adició de “NAcetyl cysteine”, proporcionen els millors valors de quantificació absoluta. L’anàlisi estadístic de les dades generades d’aquesta segona fase de qualificació i verificació de les proteïnes està encara inacabat, de manera que ara per ara no podem conclou-re res. En conclusió, les dos linies experimentals que aquí es presneten (ARN i proteïnes) represeten resultats prometedors. No obstant això, estudis de validació en poblacions independents (del tipus estudi multicèntric) són necessaris per acabar amb un biomarcador o un panell de biomarcadors vàlid per al diagnòstic del CP. Per altra banda, el seguiment dels pacients ja analitzats poden relevalar dades interessant, com la utilitat pronòstica d’aquests biomarcadors. Els resultats obtinguts haurien de tenir una aplicació clínica ràpida que, juntament amb els paràmetres d’screening actual (nivells sèrics de PSA i DRE), podrien ajudar en la pressa de decisions per CP.<br>The prostate gland is a walnut-sized organ of the male urinary-genital tract that is located below the bladder surrounding the urethra and in front of the rectum. The most important disease affecting prostate is prostate cancer (PCa). PCa is the most common cause of cancer death, and it is the second most common cause of cancer death among men in the Western world. The risk of developing this type of cancer during a lifetime is estimated at 1 in 6 men in the US, and the risk of death due to this disease is 1 in 36. The current diagnosis of PCa is based on a triad consisting of diagnosis, analysis of the levels of PSA (Prostate Specific Antigen) in serum, the digital rectal examination (DRE) and finally the prostate biopsy (PB). When serum PSA levels are above 4 ng/mL and / or when DRE is suspected, the urologist can estimate how likely the patient is affected by PCa and therefore decided the need to practice or not a PB, which is the gold standard for PCa diagnosis. The introduction of PSA testing in the late 80s, has resulted in an improvement of early diagnosis of PCa, at which time options for treatment are effective. However, despite this early detection, mortality PCa has not decreased significantly in the last 50 years. The main limitation of serum PSA as a tumor marker is its lack of specificity (around 30%) at the cut-off value of 4 ng/mL, and also a low negative predictive value (NPV), which results in a high rate of negative biopsies. Elevated PSA levels can also be attributed to other factors such as benign prostatic hyperplasia (BPH), prostatitis, etc. As a consequence of the current screening parameters, around 2/3 of the approximately 1,300,000 biopsies made yearly in the United States and 390,000 in Europe are unnecessary. In contrast, the false positive rate of a biopsy is about zero, although the false negative rate in the first biopsy may oscillates around 20%. As a result of their persistently elevated PSA levels, but negative biopsy results, these men undergo repeated biopsies to rule out PCa. This situation is called the “PSA dilemma”. For these reasons, PCa would benefit from the existence of new markers for screening and also a more specific diagnosis less invasive. Furthermore, an improvement in diagnosis would avoid many unnecessary biopsies and consequently a significant savings in the cost of health today. The search for new markers in PCa is an important field of work in the early detection of this cancer. Urine has been defined as a liquid biopsy of the urogenital tract, and it can provide much more information about these organs (including the prostate) than a tissue biopsy. Urine obtained after DRE can easily serve as a mirror of what is happening within the prostate. Furthermore, urine collection can be accomplished without disruption of clinical standard practice. It can also be repeated several times throughout the course of the prostatic disease. For all of these reasons, urine can serve as a potential source of prostate disease biomarkers. Nevertheless, using urine for biomarker discovery represents an important technical challenge, both in transcriptomic and proteomic approaches. Although there are some studies that focus on those approaches and biomarker discovery and identification, there still exists some controversy regarding the standardization of collection procedures, sample processing, storage and normalization. We hypothesized that the utilization of targeted genomic and proteomic techniques on urine samples from patients suspected of having PCa can provide a pattern of biomarkers able to efficiently distinguish between the presence or absence of a prostate carcinoma and, further, can help to identify clinically significant prostate cancer patients. The main objective of this study is to diagnose asymptomatic PCa by non/minimally-invasive means using RNA or Protein in urine after prostate massage, and to overcome the low specificity of PSA by the use of additional biomarkers to reduce the number of unnecessary biopsies (reduce financial costs for society, reduction in unwanted secondary effects). 1. TRANSCRIPTOMIC APPROACH: In recent years, the explosion of genomic and transcriptomic approaches have resulted in increased biomarker discovery. The recent discovery of Prostate Cancer Gene 3 (PCA3) in urine as a biomarker for the detection of PCa and studies to determine its applicability in routine diagnosis represent a significant success for the scientific community in this field. First, we wanted to characterize a new urine candidate biomarker (PSGR) to be compared with PCA3, and second, we planned to use a panel of biomarkers, in order to improve diagnostic accuracy. Finally, we proposed to better characterize the well-known biomarker PCA3 as a tool for the early detection of pre-neoplastic PCa lesions, such as High grade prostatic intraepithelial neoplasia (HGPIN). 1a) “PSGR and PCA3 as Biomarkers for the Detection of Prostate Cancer in Urine” Rigau M et al., Prostate. 2010 Dec 1;70(16):1760-7. PSGR is a member of the G-protein coupled OR family. PSGR has previously been described to be highly prostate tissue-specific and over-expressed in PCa tissue. Our aim was to test whether PSGR could also be detected by RTqPCR in urine sediment obtained after prostate massage (PM). A total of 215 urine samples were collected from consecutive patients (34% with PCa), who presented for PB due to elevated serum PSA levels (> 4 ng/mL) and/or an abnormal DRE. These samples were analyzed by RTqPCR. By univariate analysis we found that PSGR and PCA3 were significant predictors of PCa. A Reciever Operator Characteristics (ROC) curve was used to assess the outcome predictive values of the individual biomarkers. We obtained the following Area Under the Curve (AUC) values: PSGR (0.68) and PCA3 (0.66). Both markers individually overcame the AUC value for serum PSA (0.60). Finally, we combined those markers to test if a combination of both biomarkers could improve the sensitivity of PCA3 alone. By using a multivariate extension analysis, multivariate ROC (MultiROC), the outcome predictive values of the paired biomarkers were assessed. We obtained an AUC value of 0.73 for the combination of PSGR and PCA3 (PSGRvPCA3). Then, we tested whether a combination of PSGR and PCA3 could improve specificity by fixing the sensitivity at 95%. We obtained specificities of 15% (PSGR) and 17% (PCA3) for each individual marker and 34% for PSGRvPCA3. In summary, a multiplexed model that included PSGR and PCA3 improved the specificity for the detection of PCa, especially in the area of high sensitivity. This could be clinically useful for determining which patients should undergo biopsy. 1b) “A Three-Gene panel on urine increases PSA specificity in the detection of prostate cancer” Rigau et al., Prostate. 2011 Apr 25. doi: 10.1002/pros.21390. Much evidence points to the fact that a single marker may not necessarily reflect the multifactorial and heterogeneous nature of PCa. The principle that underlies the combined biomarker approach is consistent with tests offered for the detection of PCa in tissue specimens and takes into consideration the heterogeneity of cancer development based on a diagnostic profile. The combined model that results from these combinations provides overall increased sensitivity without decreasing the specificity. Following the same approach than in our previous work, we combined three biomarkers to maximize individual specificities. Prostate Specific Membrane Antigen (PSMA), another well-known PCa biomarker, was used to test whether a combination of PSGR, PCA3 and PSMA was able to improve the specificity of the current diagnostic technique. We analyzed post-PM urine samples from 154 consecutive patients (37% with PCa), who presented for PB due to elevated serum PSA levels (>4 ng/mL) and/or an abnormal DRE. We tested whether the putative PCa biomarkers PSMA, PSGR, and PCA3 could be detected by RTqPCR in the post-PM urine sediment. By univariate analysis, we found that the PSMA, PSGR, and PCA3 scores were significant predictors of PCa. We then combined these findings to test if a combination of these biomarkers could improve the specificity of an actual diagnosis. Using a multiplex model (PSGRvPCA3vPSMA), the area under the MultiROC curve (AUCm) was 0.74, 0.77 with PSA and 0.80 with PSA density (PSAD). Fixing the sensitivity at 96%, we obtained a specificity of 34%, 34% with PSA and 40% with PSAD. Afterwards, we specifically tested our model for clinical usefulness in the PSA diagnostic ‘‘gray zone’’ (4–10 ng/mL) on a target subset of 82 men with no prior biopsy (34% with PCa) and a target subset of 77 men with the PSAD information (35% with PCa). Using a multiplex model, the AUCm was 0.82, 0.89 with PSAD. Fixing the sensitivity at 96%, we obtained a specificity of 50% and 62% with PSAD in the gray zone. This model would allow 34% of the patients to avoid unnecessary biopsies in the gray zone (42% when using PSAD). 1c) “Behavior of PCA3 gene in the urine of men with high grade prostatic intraepithelial neoplasia” Morote and Rigau et al., World J Urol. 2010 Dec;28(6):677-80. An ideal biomarker for the early detection of PCa should also differentiate between men with isolated HGPIN and those with PCa. PCA3 is a highly specific PCa gene, and its score in post-PM urine seems to be useful in ruling out PCa, especially after a negative PB. The biopsy finding of an HGPIN is a frequent indication that the PB should be repeated. The aim of this study was to determine the efficacy of post-PM urine PCA3 scores for ruling out PCa in men with previous HGPIN. The PCA3 score was assessed by RTqPCR in 244 post- PM urine samples collected from men subjected to PB (64-isolated HGPIN, 83-PCa, and 97-benign pathology findings (BP)). The median PCA3 score was 1.56 in men with BP, 2.01 in men with isolated HGPIN and 9.06 in men with PCa. A significant difference was observed among the three scores (p < 0.001) and also between HGPIN and PCa (p = 0.008); however, no differences were observed between HGPIN and BP (p = 0.128). The AUC in the ROC analysis was 0.71 in the subset of men with BP and PCa, while it decreased to 0.63 when only men with isolated HGPIN and PCa were included in the analysis. Finally, the median of the PCA3 scores was assessed in men with previously diagnosed unifocal HGPIN (2.63) and in men with previously diagnosed multifocal HGPIN (1.59). No differences were observed between unifocal and multifocal HGPIN (p = 0.56). In conclusion, the efficacy of post-PM urine PCA3 scores in ruling out PCa in men with HGPIN is less than in men with BP. For this reason, when HGPIN is found at PB, these results should be taken into consideration, in order to establish the clinical usefulness of the PCA3 score as a tool for avoiding unnecessary repeated biopsies. 2. PROTEOMIC APPROACH: The high-throughput proteomic analysis of urine samples has recently become a popular approach for the identification of novel biomarkers. Proteins secreted by cancer cells, also referred to as "cancer cell secretomes," are a promising source for biomarker discovery. A great advantage to these cancer-secreted proteins and/or their fragments is that in most cases, they enter body fluids, such as blood or urine, and therefore, can be measured via non-invasive assays. Since the protein products of PCa cells can be detected in urine, their use as a proximal body fluid in the detection of PCa is very attractive. 2a.“The Discovery and Qualification of a Panel of Urine Biomarkers for Prostate Cancer Diagnosis”. In this study we used DIGE proteomic analysis on 30 age-matched, post-PM urine supernatant specimens, in order to identify the differentially expressed proteins in patients with PCa. 24 potential biomarkers were identified, the majority of which were secreted proteins associated with several wellknown, functional cancer pathways. Qualification of 15 of the 24 identified biomarker candidates was then undertaken by relative quantification using an SRM-based assay (target) on 50 post-PM urine supernatant samples (38% with PCa). After statistical analysis, 7 peptides, corresponding to 5 different proteins, were selected. A multiplex ROC curve using those 7 peptides showed an AUC value of 0.93. Fixing the sensitivity at 95%, we achieved a specificity of 78%. 2b. “Qualification and Verification of Urine PCa Candidate Biomarkers with Selected Reaction Monitoring”. The qualification and verification of candidate biomarkers is a critical stage in the great biomarker discovery pipeline. Credentialed biomarkers that have successfully passed through this stage are considered verified biomarkers, which are of high value for translation into large-scale, clinical validation studies. The evaluation of biomarkers in body fluids necessitates the development of robust methods to quantify proteins in body fluids, using large sets of samples. In the present study, we performed the qualification of a set of 42 candidate biomarkers for PCa diagnosis on a set of 107 post- PM urine supernatant samples (36% with PCa) using SRM-based absolute quantification. Before that, urine sample preparation and analytical procedures were optimized for SRM methodology. We standardized preparation of the urine protein samples for SRM analysis by using 9 different protocols. Our final goal was to obtain a panel of biomarkers that alone, or in combination with the existing PCa biomarker, would help us to better define patients with PCa. In addition, due to the large number of samples and their pathological conditions, we would also be able to define candidate prognostic markers. However, this study has yet to be completed. In conclusion, the data presented in this dissertation represent a significant advance in the standard care for PCa diagnosis. Our two approaches (RNA- and Protein-based) have begun to yield promising results, as both have levels of specificity that exceed those of PSA. However, validation studies on larger, multi-centric cohorts of urine samples are needed to end up with a valid PCa biomarker. The obtained results should have a rapid application in the clinics and potentially influence, together with actual screening parameters (serum PSA and DRE), decisions that could improve the health system, as well as clinical, managerial and/or public practices for health outcomes in PCa.

This thesis presents biophysical studies of new optoelecronically active DNA-binders. Chapter one gives a brief overview of the importance of DNA in medicine, of DNA structure and of the mode of interactions of small molecules with double-stranded DNA, including electrostatic, intercalation and groove interactions. Various examples of small-molecule binding to DNA are discussed. Additionally, this chapter briefly describes the biophysical techniques which can be exploited to quantify the interaction between small-molecules and duplex DNA. Chapter two describes the results of studies of the interactions of a group of 1,8-naphthalimide derivatives with double-stranded DNA using a variety of techniques viz. spectroscopy, calorimetry, viscosity and molecular docking studies. Additionally, this chapter also presents sequence selectivity studies of this group of compounds for specific sequences (dAdT)12●(dAdT)12 and (dGdC)12●(dGdC)12 through UV-visible spectroscopy. The 1,8-napthtalimide unit is shown to be a useful element for inducing DNA-binding. Chapter three describes studies of the interactions of a family of dendrimeric compounds with double-stranded DNA, again using spectroscopy, calorimetry, viscosity and molecular docking studies. Furthermore, this chapter includes sequence selectivity studies of this group of compounds for (dAdT)12●(dAdT)12 and (dGdC)12●(dGdC)12 via UV-visible spectroscopy. The charge and the length of the dendritic structures is shown to strongly affect nucleic acid affinities of this series of molecules. Chapter four describes the results of studies of the interactions of miscellaneous compounds with double-stranded DNA using variety of techniques viz. spectroscopy, calorimetry, viscosity and molecular docking studies. In addition, this chapter displays sequence selectivity studies of this group of compounds for specific sequences (dAdT)12●(dAdT)12 and (dGdC)12●(dGdC)12 via UV-visible spectroscopy. Chapter five gives an overview and general conclusions about the DNA binding studies presented in Chapters 2, 3 & 4 and finishes with suggestions for future work.

The analysis and development of robust sensing platforms based on solidly-mounted ZnO bulk acoustic wave devices was proposed. The exploitation of acoustic energy trapping was investigated and demonstrated as a method to define active sensing areas on a substrate. In addition, a new "hybrid" acoustic mode experiencing acoustic energy trapping was studied theoretically and experimentally. This mode was used as an explanation of historical inconsistencies in observed thickness-shear mode velocities. Initial theoretical and experimental results suggest that this mode is a coupling of thickness-shear and longitudinal particle displacements and, as such, may offer more mechanical and/or structural information about a sample under test. Device development was taken another step further and multi-mode ZnO resonators operating in the thickness-shear, hybrid, and longitudinal modes were introduced. These devices were characterized with respect to sample viscosity and conductivity and preliminary results show that, with further development, the multi-mode resonators provide significantly more information about a sample than their single-mode counterparts. An alternative to resonator-based platforms was also presented in the form of bulk acoustic delay lines. Initial conceptual and simulation results show that these devices provide a different perspective of typical sensing modalities by using properly designed input pulses, device tuning, and examining overall input and output signal spectra.

En l’actualitat, existeix demanda de biomarcadors per poder millorar la detecció de malalties en una etapa inicial, el seguiment de pacients i dissenyar estratègies terapèutiques. Els exosomes són partícules biològiques nanomètriques d’origen endocític, alliberades per tota classe de cèl·lules. Són vesícules portadores de molècules actives a les cèl·lules proximals i distals com a mecanisme de comunicació fisiològica, per mantenir l’homeòstasi natural i les respostes patològiques. Tots els tipus de cèl·lules fan servir exosomes per a aquest propòsit. És important destacar que una de les característiques més notables és que estan presents en tots els fluids biològics, com sang, saliva, orina, entre d’altres. La seva fàcil accessibilitat és una de les raons més convincents per a l’ús d’exosomes com biomarcadors clínics. Una altra característica sorprenent és la seva càrrega molecular, que pot ser útil per al diagnòstic i el pronòstic de diverses afeccions i malalties. Durant la biogènesi, els components de la cèl·lula romanen en els exosomes, incloses les tetraspaninas (CD9, CD63, CD81), proteïnes de membrana, lípids i diferents espècies d’ARN (ARNm i microARN) i ADN. Aquesta càrrega representa una signatura específica sobre el seu origen cel·lular i conté informació crítica sobre els processos que tenen lloc en diferents àrees del cos. Tot i que els exosomes es consideren candidats prometedors com a biomarcadors per millorar els mètodes de diagnòstic clínic actuals i desenvolupar proves ràpides, un dels principals inconvenients és que han de detectar-se a baixa concentració en mostres molt complexes. Els procediments convencionals per a la detecció d’exosomes generalment requereixen volums de mostra relativament grans i impliquen etapes preliminars de purificació i preconcentració per ultracentrifugació. Aquesta tesi aborda un dels principals problemes per simplificar el procediment analític en la detecció d’exosomes: el desenvolupament de nous mètodes de separació en fase sòlida per evitar la ultracentrifugació. Aquesta tesi aborda l’aïllament específic dels exosomes mitjançant partícules magnètiques modificades amb anticossos específics, que pot combinar-se fàcilment amb tecnologies emergents per a la detecció ràpida d’exosomes. Així mateix, es presenta un estudi exhaustiu de les proteïnes de superfície en exosomes, que poden ser reconegudes per partícules magnètiques. Aquest estudi es realitza inicialment per mètodes clàssics, inclòs l’aïllament per ultracentrifugació i la caracterització per microscòpia electrònica de transmissió, anàlisi de seguiment de nanopartícules, microscòpia confocal i detecció de proteïnes de superfície per citometria de flux. Es presenta un estudi comparatiu del perfil de biomarcadors de les cèl·lules i els seus exosomes derivats, incloses les tetraspaninas generals CD9, CD63 i CD81, i els receptors específics relacionats amb el càncer (CD24, CD44, CD54, CD326 i CD340). Segons aquest estudi, els exosomes es preconcentran per separació immunomagnètica en partícules magnètiques modificades amb antiCD81 per aconseguir més senyal en la detecció espectrofotomètrica i en biosensors de transducció electroquímica. Després s’avalua l’efecte de la matriu del sèrum sobre la separació immunomagnètica. Finalment, l’estudi d’exosomes per a la monitorització de càncer de mama també s’aborda en aquesta tesi doctoral, mitjançant diferents mètodes de diagnòstic en diferents formats, inclosos immunoassaigs i biosensors electroquímics, per millorar el rendiment analític i simplificar el procediment. Totes les estratègies presentades aquí discriminen pacients sans i amb càncer de mama en funció de biomarcadors específics relacionats amb el càncer epitelial. D’aquesta tesi es pot concloure que una major quantitat en el sèrum d’exosomes que expressen patrons moleculars de cèl·lules epitelials, així com l’activitat de fosfatasa alcalina, és un biomarcador prometedor en el càncer de mama.<br>Existe en la actualidad una demanda creciente de biomarcadores que pueden mejorar la detección de enfermedades en una etapa temprana, así como para el seguimiento de pacientes y estrategias terapéuticas. Los exosomas podrían ser los próximos candidatos para conseguir este objetivo. Son partículas biológicas nanométricas de origen endocítico, que se liberan por las células. Llevan su carga de moléculas activas a otras células proximales del cuerpo como mecanismo de comunicación fisiológica. Es importante destacar que una de las características más notables es que están presentes en todos los fluidos biológicos, como sangre, saliva, orina, entre otros. Su fácil accesibilidad es una de las razones más convincentes para el uso de exosomas como biomarcadores clínicos. Otra característica sorprendente es su carga molecular, que puede ser útil para el diagnóstico y el pronóstico de varias afecciones y enfermedades. Durante la biogénesis, los componentes de la célula permanecen en los exosomas, incluidas las tetraspaninas (CD9, CD63, CD81), proteínas de membrana, lípidos y diferentes especies de ARN (ARNm y microARN) y ADN. Esta carga representa una firma específica sobre su origen celular y contiene información crítica sobre los procesos que ocurren en diferentes áreas del cuerpo. Aunque los exosomas se consideran candidatos prometedores como biomarcadores para mejorar los métodos de diagnóstico clínico actuales y desarrollar pruebas rápidas, uno de los principales inconvenientes es que deben detectarse a baja concentración en muestras muy complejas. Los procedimientos convencionales para la detección de exosomas generalmente requieren volúmenes de muestra relativamente grandes e implican etapas preliminares de purificación y preconcentración por ultracentrifugación. Esta tesis aborda uno de los cuellos de botella que deberían considerarse para simplificar el procedimiento analítico en la detección de exosomas: el estudio y el desarrollo de nuevos métodos de separación en fase sólida para evitar la ultracentrifugación. Esta tesis aborda el aislamiento específico de los exosomas mediante partículas magnéticas, que puede combinarse fácilmente con tecnologías emergentes para la detección rápida. Asimismo, se presenta un estudio exhaustivo de las proteínas de superficie en exosomas, que pueden ser reconocidas por partículas magnéticas. Este estudio se realiza inicialmente por métodos clásicos, incluido el aislamiento por ultracentrifugación y la caracterización por microscopía electrónica de transmisión, análisis de seguimiento de nanopartículas, microscopía confocal y citometría de flujo. Se presenta un estudio comparativo del perfil de biomarcadores de las células y sus exosomas derivados, incluidas las tetraspaninas generales CD9, CD63 y CD81, y los receptores específicos relacionados con el cáncer (CD24, CD44, CD54, CD326 y CD340). Según este estudio, los exosomas se preconcentran por separación inmunomagnética en partículas magnéticas modificadas con antiCD81 para lograr una mayor señal en la detección espectrofotométrica y en biosensores de transducción electroquímica. Luego se evalúa el efecto de la matriz del suero sobre la separación inmunomagnética. Finalmente, el estudio de exosomas para la detección de cáncer de mama también se aborda en esta tesis doctoral, mediante diferentes métodos de diagnóstico en diferentes formatos, incluidos inmunoensayos y biosensores electroquímicos, para mejorar el rendimiento analítico y simplificar el procedimiento. Todas las estrategias presentadas son capaces de discriminar a pacientes sanos y con cáncer de mama en función de biomarcadores específicos relacionados con el cáncer epitelial. De esta tesis se puede concluir que una mayor cantidad en el suero de exosomas que expresan patrones moleculares de células epiteliales, así como la actividad de fosfatasa alcalina, es un biomarcador prometedor para pacientes con cáncer de mama.<br>There is a growing demand for biomarkers that can help detect diseases at an early stage, as well as for follow-up of patients and therapeutic strategies. Exosomes could be the next big step to reach this goal. Exosomes are membrane encapsulated biological nanometric particles of endocytic origin, which are released by all types of cells. They carry a cargo of active molecules to proximal and distal cells of the body as a mechanism of physiological communication, to maintain natural homeostasis as well as pathological responses. All types of cells use exosomes for this purpose. Importantly, one of the most remarkable features is that they are present in all the biological fluids, such as blood, saliva, urine, among others. Their easy accessibility is one of the most compelling reasons for developing exosomes as clinical biomarkers. Another striking characteristic is their molecular cargo, which can be useful for diagnosis and prognosis of several conditions and diseases. During the biogenesis, components of the cell remain in the exosomes, including tetraspanins (CD9, CD63, CD81), membrane proteins, lipids and different RNA species (mRNA and microRNA) and DNA. This cargo provides a specific signature about their cellular origin and contains critical information about processes happening at different areas of the body. Although the exosomes are considered promising candidates as biomarkers to improve the current clinical diagnostic methods and to develop rapid tests, one of the main drawbacks is that they must be detected at low concentration in very complex samples. Accordingly, conventional procedures for exosome detection usually require relatively large sample volumes and involve preliminary purification and preconcentration steps by ultracentrifugation. Therefore, this thesis addresses one of the bottlenecks that should be considered to simplify the analytical procedure in the detection of exosomes: the study and development of novel solid-phase separation methods in order to avoid ultracentrifugation. This thesis studies the specific isolation of exosomes on particle-based magnetic enrichment, which can be easily coupled with emerging technologies for the rapid detection of exosomes. A rational study of the surface proteins in exosomes, which can be recognized by magnetic particles is presented. This study is initially performed by classical methods, including isolation by ultracentrifugation, and characterization by Transmission Electron Microscopy, Nanoparticle Tracking Analysis, confocal microscopy and surface protein screening by Flow Cytometry. A comparative study of the biomarker profiling of the cells and their derived exosomes is discussed, including the general tetraspanins CD9, CD63 and CD81, and the specific cancer-related receptors (CD24, CD44, CD54, CD326 and CD340). Based on this study, the exosomes are preconcentrated by immunomagnetic separation on antiCD81-modified magnetic particles in order to achieve further detection based on spectrophotometric readout and electrochemical biosensing. The effect of the serum matrix on the immunomagnetic separation is then carefully evaluated. Finally, the study of exosomes for the detection of breast cancer is also addressed in this doctoral thesis, by different diagnostic methods in different formats, including immunoassays and electrochemical biosensors, in order to improve the analytical performance and simplified the procedure. All the strategies presented here are able to discriminate healthy and breast cancer patients based on specific epithelial cancer-related biomarkers. From this dissertation it can be concluded that an increased amount in the serum of exosomes expressing epithelial cells molecular patterns as well as alkaline phosphatase activity is a promising biomarker for breast cancer patients.

Luminescent transition metal complexes have arisen as viable alternatives to organic dyes for sensory applications due to their notable advantages. This thesis aimed to synthesize different kinds of iridium(III) complexes, explore their interactions with DNAs and investigate their application for the construction of oligonucleotide-based sensing platforms for important biomarkers. A series of iridium(III) complexes incorporating a variety of C^N and N^N donor ligands were synthesized and were demonstrated to possess G-quadruplex-selective binding properties via emission titration, UV/vis titration, fluorescence resonance energy transfer melting and G-quadruplex fluorescent intercalator displacement experiments. These G-quadruplex-selective iridium(III) complexes were utilized as signal transducers to monitor the conformational changes of oligonucleotides in oligonucleotide-based luminescent detection platforms for protein tyrosine kinase-7, interferon-gamma, sialic acidbinding immunoglobulin-likelectin-5 and thymine DNA glycosylase. And these designed platforms could work effectively in the diluted cell extract as the results in this thesis indicated.

Pancreatic cancer is one of the deadliest cancers in the world. The main reason is that patients do not show symptoms until the advanced stages of the disease, when it has spread to other organs. In the late stages, there is no cure for pancreatic cancer and the only feasible treatments are chemotherapy and radiotherapy, which only prolong survival by a few months. In fact, only 15% of patients present when the disease is still resectable with surgery, which is the only cure. The main reason for these poor outcomes is that there is no diagnostic test to detect the disease in the early asymptomatic stage. Nanotechnology is an emerging field that is providing solutions to some of the dilemmas in cancer. For diagnostics, nanoparticles are being used to increase the sensitivity of detecting circulating cancer biomarkers in blood using a phenomenon known as metal enhanced fluorescence. By increasing the sensitivity of detecting these biomarkers, it has been shown that lower tumour burden can be identified. Furthermore, novel biomarkers such as microRNAs are proving to be promising candidates as biomarkers for early pancreatic cancer. In this project, four different nanomaterials for metal enhanced fluorescence-based detection of biomarkers were developed and evaluated using pancreatic cancer biomarkers. The nanomaterials have been optimised for use in biosensing. The lowest limit of detection achieved using tunable gold nanotriangular arrays was 7.7 × 10−7 UmL−1 and 10-15 M for CA 19–9 and GPC-1 respectively. A concentration dependent metal enhanced fluorescent assay was achieved for microRNA-21, also for detecting pancreatic cancer. Both assays were highly specific for their respective analyte of interest. These nanomaterials show great potential as multiplexing platforms to simultaneously detect multiple biomarkers for pancreatic cancer. In future, this strategy may improve the detection of the disease in the early stages to improve patient outcomes.

Thesis (Ph.D.)--Boston University<br>Label-free optical biosensors have been established as proven tools for monitoring specific biomolecular interactions. However, compact and robust embodiments of such instruments have yet to be introduced in order to provide sensitive, quantitative, and high-throughput biosensing for low-cost research and clinical applications. Here we present the interferometric reflectance-imaging sensor (IRIS). IRIS allows sensitive label free analysis using an inexpensive and durable multi-color LED illumination source on a silicon based surface. IRIS monitors biomolecular interaction through measurement of biomass addition to the sensor's surface. We demonstrate the capability of this system to dynamically monitor antigen-antibody interactions with a noise floor of 5.2 pg/mm^2 and DNA single mismatch detection under isothermal melting conditions in an array format. Ensemble detection of binding events using IRIS did not provide the sensitivity needed for detection of infectious disease and biomarkers at clinically relevant concentrations. Therefore, a new approach was adapted to the IRIS platform that allowed the detection and identification of individual nanoparticles on the sensor's surface. The new detection method was te1med single-particle IRIS (SP-IRIS). We developed two detection modalities for SP-IRIS. The first modality is when the target is a nanoparticle such as a virus. We verified that SP-IRIS can accurately detect and size individual viral particles. Then we demonstrated that single nanoparticle counting and sizing methodology on SP-IRIS leads to a specific and sensitive virus sensor that can be multiplexed. Finally, we developed an assay for the detection of Ebola and Marburg. A detection limit of 3 x 10^3 PFU/ml was demonstrated for vesicular stomatitis virus (VSV) pseudotyped with Ebola or Marburg virus glycoprotein. We have demonstrated that virus detection can be done in human whole blood directly without the need for sample preparation. The second modality of SP-IRIS we developed was single molecule counting of biomarkers utilizing a sandwich assay with detection probes labeled with gold nanoparticles. We demonstrated the use of single molecule counting in a nucleic acid assay for melanoma biomarker detection. We showed that a single molecule counting assay can lead to detection limits in the attomolar range. The improved sensitivity of IRIS utilizing single nanoparticle detection holds promise for a simple and low-cost technology for rapid virus detection and multiplexed molecular screening for clinical applications.

Extracellular vesicles (EVs) are small membrane-surrounded structures containing transmembrane proteins and enclosing cytosolic proteins and nucleic acids. They are released in the extracellular space by both normal and neoplastic cells and play an important role in cell-cell communication in numerous physiological processes and pathological conditions, through the transfer of their functional cargo to recipient cells. EVs are highly abundant in biological fluids, and even more represented in cancer patients’ biofluids, therefore many studies suggested that they can be instrumental in liquid biopsies as prognostic markers or for early detection of tumors. Moreover, being secreted by potentially all the cells, they can serve in oncology to represent the tumor heterogeneity, which is underestimated by the current diagnostic tools. Given their small size, EVs are difficult to isolate in a high-throughput way and, therefore, one of the main obstacles to their clinical application, is that the existing isolation methods are impractical. During these years, I worked at the development and optimization of a novel technique that allows purification of heterogeneous EVs from biological fluids in an efficient, fast and reproducible way. This technique, named Nickel-Based Isolation (NBI), is a biochemical assay that allows obtaining polydisperse EVs in a physiological pH solution, therefore, preserving their morphology, heterogeneity, and stability. We tested and optimized this assay in protein-enriched systems and comparing it to the techniques currently used to characterize and measure EVs, such as flow cytometry and Tunable Resistive Pulse Sensing. We challenged the reproducibility of this method by isolating EVs from different biological fluids. Interestingly, the EVs purified with NBI result more intact and stable compared to the ones obtained with other methods, and can be studied in a clinical setting and used as an innovative tool for detection of molecules associated with diseases. We demonstrated the specificity of the procedure by using individual isolated vesicles in biochemical and molecular assay, optimized to characterize the biological content of EVs. We were able to detect picomolar concentration of PSMA on 105 EVs isolated from plasma of prostate cancer patients and BRAF-V600E transcript in just 103 EVs from the plasma of colon cancer patients, reaching unprecedented matching with tissue biopsy results. We also investigated the transcriptome of EVs isolated from glioblastoma cancer stem cells, in order to exploit the potential of EVs as diagnostic markers.

Thesis advisor: Thomas C. Chiles<br>Rapid diagnosis of infectious disease at the site of the patient is critical for preventing the escalation of an outbreak into an epidemic. This is particularly true for cholera, a disease known to spread swiftly within resource-limited populations. A device suited to point-of- care (POC) diagnosis of cholera must not only demonstrate laboratory levels of sensitivity and specificity, but it must do so in a highly portable, low-cost manner, with a simplistic readout. Here, we report novel proof-of-concept lab-on-a-chip (LOC) electrochemical immunosensors for the detection of cholera toxin subunit B (CTX), based on two nanostructured architectures: the gold dendritic array, and the extended core coax (ECC). The dendritic array has an ~18x greater surface area than a planar gold counterpart, per electrochemical measurements, allowing for a higher level of diagnostic sensitivity. An electrochemical enzyme-linked immunosorbant assay (ELISA) for CTX performed via differential pulse voltammetry (DPV) on the dendritic sensor demonstrated a limit-of detection of 1 ng/mL, per a signal-to-noise ratio of 2.6, which was more sensitive than a simple planar gold electrode (100 ng/mL). This sensitivity also matches a currently available diagnostic standard, the optical ELISA, but on a miniaturized platform with simple electrical readout. The ECC was optimized and explored, undergoing several changes in design to facilitate sensitive LOC electrochemical detection. The ECC matched the off-chip sensitivity towards CTX demonstrated by a previous non-extended core coaxial iteration, which was comparable to a standard optical ELISA. In contrast to the previous coaxial architecture, the ECC is amenable to functionalization of the gold core, allowing for LOC detection. ECCs were functionalized using a thiolated protein G, and CTX was detected via an electrochemical ELISA. While this work is ongoing, the ECC shows promise as a platform for LOC electrochemical ELISA. The ability to potentially meet POC demands makes biofunctionalized gold dendrites and ECCs promising architectures for further development as LOC sensors for the detection of infectious disease biomarkers<br>Thesis (PhD) — Boston College, 2018<br>Submitted to: Boston College. Graduate School of Arts and Sciences<br>Discipline: Biology

The Proximity Ligation Assay (PLA) is a relatively new method which utilizes the strength of both immunoassays and DNA detection. PLA has the capacity of high multiplexing due to the high specificity achieved with both dual protein-binding and dual primer binding during detection with Real-Time PCR. We developed a multiplexed PLA protocol that can measure 28 biomarkers in human EDTA plasma. The method was tested on 46 individuals diagnosed with colorectal cancer and 48 age matched healthy controls. The results are very promising as we re-discover the most well-known biomarkers for colorectal cancer and also find some potential new markers (significance tested with students T-test with p&lt;0.05). Further improvements of the protocol are needed to decrease the variation.

La maladie d’Alzheimer est la première cause de démence chez les personnes âgées. Cette maladie est caractérisée par un déclin irréversible des fonctions cognitives. Les patients atteints par la maladie d’Alzheimer ont de sévères pertes de mémoire et ont de grandes difficultés à apprendre de nouvelles informations ce qui pose de gros problèmes dans leur vie quotidienne. À ce jour, cette maladie est diagnostiquée après que d’importantes altérations des structures du cerveaux apparaissent. De plus, aucune thérapie existe permettant de faire reculer ou de stopper la maladie. Le développement de nouvelles méthodes permettant la détection précoce de cette maladie est ainsi nécessaire. En effet, une détection précoce permettrait une meilleure prise en charge des patients atteints de cette maladie ainsi qu’une accélération de la recherche thérapeutique. Nos travaux de recherche portent sur l’utilisation de l’imagerie médicale, avec notamment l’imagerie par résonance magnétique (IRM) qui a démontrée ces dernières années son potentiel pour améliorer la détection et la prédiction de la maladie d’Alzheimer. Afin d’exploiter pleinement ce type d’imagerie, de nombreuses méthodes ont été proposées récemment. Au cours de nos recherches, nous nous sommes intéressés à un type de méthode en particulier qui est basé sur la correspondance de patchs dans de grandes bibliothèques d’images. Nous avons étudié ces méthodes à diverses échelles anatomiques c’est à dire, cerveaux entier, hippocampe, sous-champs de l’hippocampe) avec diverses modalités d’IRM (par exemple, IRM anatomique et imagerie de diffusion). Nous avons amélioré les performances de détection dans les stades les plus précoces avec l’imagerie par diffusion. Nous avons aussi proposé un nouveau schéma de fusion pour combiner IRM anatomique et imagerie de diffusion. De plus, nous avons montré que la correspondance de patchs était améliorée par l’utilisation de filtres dérivatifs. Enfin, nous avons proposé une méthode par graphe permettant de combiner les informations de similarité inter-sujet avec les informations apportées par la variabilité intra-sujet. Les résultats des expériences menées dans cette thèse ont montrées une amélioration des performances de diagnostique et de prognostique de la maladie d’Alzheimer comparé aux méthodes de l’état de l’art<br>Alzheimer’s disease (AD) is the most common dementia leading to a neurodegenerative process and causing mental dysfunctions. According to the world health organization, the number of patients having AD will double in 20 years. Neuroimaging studies performed on AD patients revealed that structural brain alterations are advanced when the diagnosis is established. Indeed, the clinical symptoms of AD are preceded by brain changes. This stresses the need to develop new biomarkers to detect the first stages of the disease. The development of such biomarkers can make easier the design of clinical trials and therefore accelerate the development of new therapies. Over the past decades, the improvement of magnetic resonance imaging (MRI) has led to the development of new imaging biomarkers. Such biomarkers demonstrated their relevance for computer-aided diagnosis but have shown limited performances for AD prognosis. Recently, advanced biomarkers were proposed toimprove computer-aided prognosis. Among them, patch-based grading methods demonstrated competitive results to detect subtle modifications at the earliest stages of AD. Such methods have shown their ability to predict AD several years before the conversion to dementia. For these reasons, we have had a particular interest in patch-based grading methods. First, we studied patch-based grading methods for different anatomical scales (i.e., whole brain, hippocampus, and hippocampal subfields). We adapted patch-based grading method to different MRI modalities (i.e., anatomical MRI and diffusion-weighted MRI) and developed an adaptive fusion scheme. Then, we showed that patch comparisons are improved with the use of multi-directional derivative features. Finally, we proposed a new method based on a graph modeling that enables to combine information from inter-subjects’ similarities and intra-subjects’ variability. The conducted experiments demonstrate that our proposed method enable an improvement of AD detection and prediction

The detection of cancer biomarkers is of great importance in oncology. Cancer biomarkers can provide diagnostic information which can aid disease screening and early diagnosis. Further, cancer biomarkers can help predict disease prognosis and response to therapy, and also help in the monitoring of disease. Thence, the accurate and sensitive detection of cancer biomarkers which may be present at very low concentrations is of great clinical importance. Traditionally, these biomarkers have been detected predominantly by Enzyme Linked Immunosorbent Assay. The traditional biomarker detection assays generally require multiple washing steps, long assay times, and have the need for trained expertise and expensive instrumentation. In this dissertation, Lateral Flow Strip Biosensors (LFSB) that provide rapid, low-cost and user-friendly screening of cancer biomarkers are discussed. The developed biosensors have the added advantages of being portable, sensitive and highly selective, which makes them ideal for routine cancer screening. Gold nanoparticles (GNP)-based Lateral Flow Strip Immunosensors (LFSI) that colorimetrically detected carbohydrate antigen (CA 19-9) and carcinoembryonic antigen (CEA) were developed for the screening of human plasma and pancreatic cyst fluid, respectively. Further, carbon nanotube based-LFSBs that targeted CA 19-9 and CEA were developed. The CNT-based LFSBs showed improved detection limits over the conventional GNP-based LFSB. A GNP-based LFSB was also developed for the detection of exosomes using an aptamer that targeted a cell surface protein, epithelial cell adhesion molecule (EpCAM). The developed assays showed good performance and were used for the screening of pancreatic cancer patient samples. Upon further development, the assays discussed in this dissertation could find application in the clinical screening and monitoring of cancer, especially in limited resource settings.<br>National Institutes of Health (U.S.)<br>NIH-COBRE (1P20GM09024)<br>North Dakota State University. Department of Chemistry and Biochemistry

Background: Oral squamous cell carcinoma (OSCC) is the 15th most common cancer worldwide but has poor five year survival (50%). Late stage presentation and limitations of early diagnostic techniques are persistent clinical problems. Sixty percent of patients present with advanced stage disease and with the attendant increase in mortality, morbidity and risk of recurrent disease it is particularly burdensome for both patients and health economies. Early diagnosis and treatment of OSCC improves prognosis. There is an opportunity to diagnose OSCC early in patients with oral epithelial dysplasia however currently there is no way of accurately predicting which lesions will undergo malignant transformation. Aberrant methylation of tumour suppressor genes plays a significant role in the biology of early cancer and is detectable in both tumour and saliva. Saliva is a non-invasive method of longitudinal sampling and has potential as a tumour surrogate in disease surveillance programmes. This study aims to compare rates of methylation of a panel of genes in OSCC patients and a normal cohort to establish a threshold by which we could determine future disease testing in a dysplastic population. Methods: Saliva samples were collected from 219 individuals from three diagnostic groups: Normal (defined as no oral malignant or premalignant disease) n=97, OSCC n=62 and dysplasia n=60. For statistical analysis the dysplasia cohort was sub-divided into lesions of low and high risk of malignant transformation based on the histological diagnosis of the index lesion. DNA was extracted and bisulphite treated from 258 saliva samples before duplex quantitative methylation specific PCR (qMSP) assays were performed on all samples to detect the frequency of methylation in saliva of a panel of genes. The five target genes (ADAMTS9, CCNA1, CYGB, P16, TMEFF2) were selected using a candidate approach on the basis of tumour specificity from studies on tumour/normal matched tissue pairs. Clinicopathological data was correlated with the qMSP data and analysed using SPSS v.21 statistical software to look for associations with tumour and survival characteristics. Results: Only 3/97 individuals from the control normal cohort had saliva samples with detectable methylation above the analytical sensitivity of the P16 assay. Methylation of the remaining target genes (ADAMTS9, CCNA1, CYGB, TMEFF2) was not detected in normal saliva at levels above the analytical sensitivity of the qMSP assays. The most significant finding in this study was that methylation of four of the target genes (CCNA1, CYGB, P16, TMEFF2) in saliva, individually and when considered as a panel, was significantly associated with OSCC and as such could aid discrimination between malignant disease and normal saliva samples. Methylation of at least one gene in the panel was discovered in 29/67 of the binned OSCC saliva samples but only 3/97 of normal samples (Fisher’s exact p=0.001). Furthermore methylation of the gene panel is associated with high risk lesions when detected in saliva of patients with premalignant lesions (Fisher’s exact p=0.03). Conclusions: This exploratory data supports the utility of duplex qMSP as a detection method for methylation markers in saliva. The detection of methylation of this gene panel in saliva is significantly more associated with oral malignancy and high risk premalignant lesions than normal and low risk disease. This implies saliva may have merit as a surrogate tissue in an adjunctive role to clinical assessment and biopsy. The assays are specific but have limited sensitivity. However with further work, inclusive of additional genes, this methodology may identify predictive biomarkers that can be introduced into a trial surveillance of premalignant lesions.

Proteins are the effector molecules of life. They are encoded in DNA that is inherited from generation to generation, but most cellular functions are executed by proteins. Proteins rarely act on their own – most actions are carried out through an interplay of tens of proteins and other biomolecules. Here I describe how synthetic DNA can be used to study proteins and protein complexes. Variants of proximity ligation assays (PLA) are used to generate DNA reporter molecules upon proximal binding by pairs of DNA oligonucleotide-modified affinity reagents. In Paper I, a robust protocol was set up for PLA on paramagnetic microparticles, and we demonstrated that this solid phase PLA had superior performance for detecting nine candidate cancer biomarkers compared to other immunoassays. Based on the protocol described in Paper I I then developed further variants of PLA that allows detection of protein aggregates and protein interactions. I sensitively detected aggregated amyloid protofibrils of prion proteins in paper II, and in paper III I studied binary interactions between several proteins of the NFκB family. For all immunoassays the selection of high quality affinity binders represents a major challenge. I have therefore established a protocol where a large set of protein binders can be simultaneously validated to identify optimal pairs for dual recognition immunoassays (Paper IV).

Magister Scientiae - MSc<br>Breast cancer (BC) is the most common cancer in women worldwide, and is the second most common cancer in the world, responsible for more than 500 000 deaths annually. Estimates are that 1 in 8 women will develop BC in their lifetime. In South Africa, BC in women affects about 16.6 % of the population and could see a 78 % increase in cases by 2030. The failure of conventional diagnostic tools to detect BC from an early onset has revealed the need for diagnostic tools that would enable early diagnosis of BC. The current diagnostic tools include breast self-examination, mammography magnetic resonance imaging, ultrasonography and serum biomarkers; BRACA1, BRACA2, HER2. These conventional methods lack sensitivity, specificity and positive predictive value, and some of these diagnostic tools may be expensive and quite invasive. Therefore, novel diagnostic tools such as microRNAs which address the short comings of current methods are required for early diagnosis as well as BC management. MicroRNAs are a class of non-coding RNA molecules, which are important in RNA stability and gene expression. Various methodologies have been employed to identify novel microRNAs for diagnostics such as bioinformatics, also referred to as in silico analysis. The aim of this study is to identify novel microRNAs that can potentially detect BC at its earliest stage.

El cáncer de próstata (CP) es una de las mayores causas de muerte entre los hombres de los países desarrollados. Sin embargo, los actuales métodos de diagnóstico presentan una elevada tasa de falsos positivos debido a su baja especificidad, lo que conlleva un elevado número de biopsias de próstata (BPs) practicadas innecesariamente. Además, en muchas BPs, alguna patología no cancerosa de la próstata es hallada. Una patología benigna particularmente relevante en la clínica es la Neoplasia Intraepitelial Prostática de Alto Grado (HGPIN), reconocida como el precursor más probable del CP. Cuando se descubre en una BP, se asocia con una mayor probabilidad de existencia de un CP no detectado. Por ello, estos pacientes son seguidos clínicamente durante años, incluyendo la realización de múltiples BPs de repetición. Sin embargo, sólo en una pequeña parte de los pacientes un CP será finalmente encontrado. En resumen, la gran mayoría de las BPs practicadas actualmente son innecesarias, constituyendo una molestia para el paciente y un gran gasto económico para el sistema de salud. En los últimos años se ha hecho un gran esfuerzo en la identificación de nuevos biomarcadores para el CP, con el objetivo de mejorar la situación actual. Gracias a la ubicación de la próstata en el cuerpo, en contacto directo con la uretra, células descamadas y productos secretados, incluyendo exosomas, se pueden encontrar en la orina. El objetivo principal de esta tesis es la identificación de nuevos biomarcadores en orina, con el fin de desarrollar un método no invasivo para el diagnóstico precoz y correcto de CP, tanto en pacientes referidos para una primera BP como en pacientes previamente diagnosticados con HGPIN. Los exosomas han sido propuestos como una potencial nueva fuente de biomarcadores, ya que contienen moléculas que representan sus tejidos de origen. Con el fin de identificar nuevos biomarcadores CP en exosomas urinarios, en primer lugar hemos establecido un método de aislamiento de vesículas fiable, tras lo que las vesículas obtenidas fueron caracterizadas por microscopía electrónica, Western blot y nanoparticle tracking analysis. A continuación, se realizó una fase de descubrimiento mediante LC-MS/MS label-free (n = 24). El análisis de los datos se llevó a cabo mediante medición de las intensidades iónicas, aplicando varios enfoques estadísticos con el fin de aumentar la fiabilidad de los resultados. Una vez obtenida una lista de biomarcadores candidatos, éstos fueron validados siguiendo un enfoque de la proteómica dirigida (Selected Reaction Monitoring, SRM). Finalmente, se ha identificado un perfil de 15 biomarcadores expresados diferencialmente en CP. Dos de estas proteínas, que corresponden a una subunidad alfa y una beta de integrina, fueron seleccionadas para una caracterización adicional usando modelos in vitro, debido a su interés biológico. Esta caracterización demostró la localización intracelular de estos biomarcadores en endosomas tardíos. En el contexto de las BPs de repetición por diagnóstico de HGPIN, hemos investigado una serie de biomarcadores de CP basados en ARN del sedimento urinario. Como resultado, hemos descrito tres modelos multiplex con diferentes combinaciones de los genes CDH1, PSMA, PSGR y KLK3. Todos estos modelos superan a los métodos actualmente utilizados para la detección del CP en hombres con HGPIN, y su uso en la clínica podría potencialmente ahorrar una gran cantidad de BPs de repetición innecesarias. En el futuro, los resultados derivados de este estudio podrían mejorar la actual detección del CP, distinguiendo un CP agresivo de aquel clínicamente insignificante y otras condiciones benignas y, por lo tanto, evitando el sobrediagnóstico y sobretratamiento relacionados con esta enfermedad.<br>Prostate cancer (PCa) is one of the leading causes of death among men in the developed countries. Nevertheless, current methods of diagnosis for PCa have a high rate of false positive results due to a low specificity and, therefore, to many unnecessary prostatic biopsies (PBs). Moreover, in many PBs a non-cancerous pathology of the prostate is found. A particularly clinically relevant benign condition is High Grade Intraepithelial Neoplasia (HGPIN), recognized as the most probable precursor to PCa. When found in PB, it is associated with an increased probability of the existence of an undetected PCa. For this reason, its encounter in a first PB guarantees an intensive surveillance over the years, including multiple repeat PBs. However, only in a small part of these patients an aggressive form of PCa will be eventually found. In summary, the great majority of the PBs practiced nowadays are unnecessary, causing pointless discomfort to the patient and extra expenses to the health care system. In the last years, a lot of effort has been put into the identification of new biomarkers for PCa that would improve the current situation. Because of the location of the prostate in the body, in direct contact with the urethra, desquamated cells and secreted products, including exosomes, can be detected in urine. The main aim of this thesis is the identification of new biomarkers for PCa in urine, in order to develop a non-invasive method for the early and accurate diagnosis of PCa, both in a first PB setting and in patients already diagnosed with HGPIN. Exosomes have been proposed as a potential new source for biomarkers, since they contain molecules representing their tissues of origin. In order to identify new PCa biomarkers in urinary exosomes, we have first established a reliable vesicles isolation method, and then the obtained vesicles were characterized by electronic microscopy, Western blot, and nanoparticle tracking analysis. Next, we carried out a discovery phase by label-free LC-MS/MS (n=24). Analysis of the data was carried out measuring the ion peak intensities and applying several different statistic approaches, in order to increase the reliability of the results. Once we had a list of biomarker candidates, these were validated following a targeted proteomics approach (Selected Reaction Monitoring, SRM). Finally, we have identified a profile of 15 differentially expressed protein biomarkers for PCa. Two of these validated proteins, corresponding to one alpha and one beta integrin subunits, were selected for further characterization using in vitro models, based on biological interest. This characterization proved the intracellular location of these protein biomarkers in late endosomes. In the context of repeat PBs due to a previous diagnosis of HGPIN, we have assayed a set of known RNA-based PCa biomarkers from urine sediment. As a result, we have described a set of three multiplex models with different combinations of the genes CDH1, PSMA, PSGR and KLK3. All these models outperform the currently existent methods for the detection of PCa in men with HGPIN, and their use in clinical practice could potentially save a high number of unnecessary repeat PBs. In the future, the results derived from this study could improve current PCa detection, distinguishing aggressive from clinically insignificant PCa and other benign conditions and, therefore, avoiding PCa related over-diagnosis and over-treatment.

The role of DNA methylation based biomarkers in several stages of cancer development is a rapidly advancing area of research. The aberrancies on the methylation profile of gene promoters play a critical role in gene silencing, thus contributing in different phases of tumour initiation, progression and recurrence, also associated with predicting the response to chemotherapeutic agents, therefore leading to a better assessment of the clinical effectiveness of cancer therapies and so of better prognosis. The need for detection of DNA methylation has become one of the most important assays in early cancer screening. This work introduces the use of semiconductor technology for detection of DNA methylation based biomarkers in CMOS for early screening of cancer using the Ion-Sensitive Field-Effect Transistor (ISFET). This enables label-free detection of DNA methylation in gene markers of interest associated with tumour development in different organs, ultimately enabling a Point-of-Care (PoC) system for early cancer diagnosis. Towards this goal, we introduce the concept of ratiometric detection using the ``Methylation Cell" which allows continuous computation of the DNA methylation ratio. This was demonstrated through a Lab-on-Chip (LoC) system using low power current-mode translinear circuits fabricated in unmodified CMOS. Complementary to this, a novel implementation of the ``Gilbert Gain Cell" integrated with ISFET sensors was proposed, referred to as the ISFET based Chemical Gilbert Cell, a current-mode circuit for differential reaction monitoring of pH signals derived from DNA methylation reactions. The Cell achieves elimination of common non-idealities of ISFETs such as drift reduction and temperature variations, while achieving gain tunability.

Luminescent semiconductor quantum dots (QDs) are extensively researched for use in biological applications. They have unique optical and physical properties that make them excellent candidates to replace conventional organic dyes for cellular labeling, multiplexing, nucleic acid detection, and as generalized probes. The primary focus of this dissertation was to utilize quantum dots for improvement in immunoassays. Specifically, atherosclerosis biomarkers were detected simultaneously in an effort to demonstrate advances in early detection diagnostics. Quantum dot-antibody bioconjugates were prepared by encapsulation into mesoporous silica and functionalized with thiol and amine groups to enable bioconjugation. Functionalization of the mesoporous silica quantum dot composites facilitated biocompatibility for use with biological buffers in immunoassays. These bioconjugates were used in a sandwich immunoassay to detect atherosclerosis biomarkers IL-15 and MCP-1. Sandwich assays employ capture antibodies immobilized onto a well plate to bind as much of the antigen as possible. The capture antibodies increased binding by at least 4 times the amount of antigen bound to the surface of a direct detection assay. The sandwich immunoassay was able to detect 1 pg/mL of IL-15 and 50 pg/mL of MCP-1 biomarkers. Human serum albumin nanoparticles (HSAPs) were synthesized via a desolvation and crosslinking method. Human serum albumin is a versatile protein being used in a variety of applications. Quantum dots were loaded into HSAPs as potential detection probes for immunoassays. Efficient loading was not achieved, and the assay was unable to improve current detection limits. Controlled release studies were explored using HSAPs loaded with superparamagnetic iron oxide nanoparticles and a fluorescent drug analog. Exposure to a magnetic field resulted in degradation of the HSAPs. The fluorophore was released and measured to examine how cancer drugs might be controlled through a magnetic field. Gold nanorods and an anticancer drug, Sorafenib, were also encapsulated into HSAPs for treatment of renal cell carcinoma in vivo. Laser irradiation treatment combined with Sorafenib resulted in 100% tumor necrosis and total elimination of any viable tumor present. HSAPs have demonstrated remarkable potential as drug delivery nanocarriers.

There has been much interest in recent years about the potential of microRNA as a new source of biomarkers for the diagnosis of disease. The delivery of new diagnostic tools based on this potential has been limited by shortcomings in current microRNA detection techniques. This thesis explores the development of a new method of microRNA detection through the incorporation of conductive particles into oligonucleotide-functionalised polymers to form oligonucleotide cross-linked polymer composites. Such composites could provide a simple, rapid, and low-cost means of microRNA detection that could be easily multiplexed, providing a valuable tool for point-of-care medical diagnostics. This work presents oligonucleotide-functionalised carbon/polyacrylamide composites which demonstrate a selective swelling response in the presence of analyte oligonucleotide sequences and for which the electrical conductivity decreases with swelling. The composites were synthesised via UV-initiated free-radical polymerisation of carbon/- monomer mixtures upon custom electrode devices, consisting of interdigitated platinum electrodes fabricated upon a silicon substrate. The optimal cross-linker density and carbon loading concentration were determined as well as the best means of dispersing the carbon particles within the polymer. Various types of carbon particles, with differing sizes and aspect ratios, were compared and their performances as conductive additives for polymer swelling transduction evaluated. The swelling behaviour of these composites was evaluated by analysing images of composite microdroplets as they swell. The electrical characteristics of the composites were determined by measuring either the two-terminal resistance or the complex impedance of composite microdroplets on the electrode devices. Alternating and direct current measurement techniques were compared to determine the best approach for the transduction of composite swelling. The volumetric and electrical responses of oligonucleotide-functionalised carbon/polyacrylamide composites were analysed in solutions of analyte oligonucleotide and non-complementary controls. It has been demonstrated that, using carbon nanopowder composites and a direct current two-terminal resistance measurement, it is possible to differentiate between analyte and control solutions to concentrations as low as 10 nM, with single-base precision, in less than three minutes. However, the inability to detect at concentrations below this value, difficulties in differentiating between different analyte concentrations and thermal instability mean that, in their current form, oligonucleotide cross-linked polymer composites are unsuitable for the detection of circulating microRNA at clinically relevant concentrations. Potential avenues of work to address these challenges are discussed. Also presented are collaborative results for oligonucleotide-responsive polymers functionalised with morpholino nucleic acid analogues, in what is believed to be the first example of such a material. These morpholino-functionalised polymers offer significant advantages, in terms of stability and sensitivity, over their nucleic acid equivalents for bio-responsive polymer applications.

Atherosclerosis is a chronic inflammatory disease leading to plaque buildup in the major arteries. The plaques consist of cholesterol, calcium, inflammatory cells, extracellular matrix and fibrous material. Under inflammatory conditions IFN-• stimulation of human monocytes and macrophages generates reduced pteridine, 7,8-dihydroneopterin (78NP) which has been shown to be an effective cytoprotective agent to some cell types against oxidative damage by reactive oxygen species (ROS). 7,8-dihydroneopterin is oxidized to fluorescent neopterin in the presence of hypochlorite (HOCl). Although a considerable amount of work has been published on the composition of neopterin in atherosclerotic plaques, very little is known about the variation of 78NP and other oxidative biomarkers across the length of the carotid and femoral and their contribution to plaque progression, which was researched in this work. Atherosclerotic plaques excised from patients with carotid and femoral plaques were sliced into 3-5 mm sections, and each section was analyzed for concentrations of neopterin, 7,8- dihydroneopterin, •-tocopherol, TBARS, DOPA, cholesterol, dityrosine, protein carbonyls •- aminoadipic semialdehyde (AAS) and •-glutamic semialdehyde (GGS), free and esterified 7- ketocholesterol (7-KC). Cultured live plaque as a source of 7,8-dihydroneopterin and neopterin was also investigated in this study. It was shown that carotid plaques significantly vary from femoral plaques, in the levels and range of most oxidative biomarkers. Carotid plaques showed a high variation in the biomarker concentrations between plaques but also between sections of an individual plaque. Femoral plaques on the other hand showed lower amounts of biomarkers with very little variation in biomarker concentrations. High variation with pterin concentrations and other biomarkers suggests dynamic and active changes in inflammation within the plaque. Collectively, it was observed that every plaque was unique with respect to its composition and correlations between the biomarkers. Though shown to be a well-known antioxidant and a radical scavenger, there is no published literature on 7,8-dihydroneopterin’s mode of entry into and out of the cell. To understand how it enters the cells could explain the difference in its protective ability of different cell types Abstract xxviii against oxidative stress-mediated cell death. Knowledge of transport of 7,8-dihydroneopterin will provide insights about its protection of monocyte/macrophage cell death which could potentially reduce atherosclerotic plaque growth and progression. As 7,8-dihydroneopterin is produced from guanosine, a nucleoside that is transported using specialized nucleoside transporters (equilibrative nucleoside transporters (ENT's) and concentrative nucleoside transporters (CNT's), their role was examined and characterized for 7,8-dihydroneopterin transport. It was found that 7,8-dihydroneopterin and neopterin are transported via nucleoside transporters in U937 cells, THP-1 cells and human monocytes. ENT 2 was the major transporter in U937 cells while ENT 1 transported bulk of 7,8-dihydroneopterin in THP-1 cells. Both ENT's and CNT's are involved in 7,8-dihydroneopterin uptake in human monocytes. In all the cell lines tested, 7,8-dihydroneopterin protection against AAPH mediated oxidative cell death was inhibited by nucleoside transport inhibitors, suggesting that nucleoside transporters are indispensible for 7,8-dihydroneopterin mediated intracellular protection against oxidative stress. Accurate measurement of neopterin, as a biomarker of inflammation in plaques and cells is critical aspect to assess disease progression. The current C18 HPLC method used in our laboratory for neopterin measurement lacks sensitivity due to interference of acetonitrile (ACN) over time. Acidic tri-iodide conversion of 7,8-dihydroneopterin to neopterin was also variable at times giving inconsistent measurement of neopterin so the manganese oxide (MnO2) method was looked at as an alternative. Electrochemical detector (ECD) was another option studied as it did not require any precolumn oxidation of 7,8-dihydroneopterin to neopterin. A new method using strong cation exchange (SCX) column was developed for a precise, sensitive neopterin assay which got rid of the ACN interference completely. The MnO2 method of 7,8-dihydroneopterin oxidation did not work with biological samples such as serum or plaque homogenates. Electrochemical detection was also found to be very unreliable and inconsistent.

Electronic biosensors utilizing micron-scale interdigitate electrodes (IDEs) in an SD card format have been developed with the objective of fast, sensitive detection of ovarian cancer biomarkers CA-125, CEA, and He4. The signal generated by the biosensors is a result of electrochemical impedance spectroscopy (EIS), a technique which probes changes that occur in the biosensor's electrical properties when the biosensor has detected one of the target biomarkers. A label-free biosensor has been developed to detect CA-125 in spiked buffer at concentrations between 10 and 80units/mL. A similar label-free biosensor was developed to detect CEA at concentrations between 10ug/mL and 10mg/mL. A biosensor employing a protein-enzyme conjugated label was developed to detect He4 at concentrations ranging from 1.56 to 100ng/mL in spiked buffer. All concentration ranges of CA-125, CEA, and He4 detected by the biosensors include the serum concentration currently used for clinical diagnosis of ovarian cancer. Efforts to improve the signals generated by the biosensors included altering the dimensions and composition of the IDEs used in the initial biosensors through software-created models. Modeled alterations included the size of the electrodes, the shape of the electrodes, and the incorporation of nanomaterials into the IDEs. An ideal geometry for the IDEs was developed through the models and IDEs with those dimensions were fabricated and tested against the IDEs used in the biosensors initially with the model-developed geometry improving the signal generated by the biosensor. Another attempt to improve the biosensor's signal was to generate a single strand of DNA (ssDNA) that would bind to CA-125, called an aptamer, that could be easily incorporated into the sensing layer on the IDEs. Through a multistep selection process nine different aptamers that exhibited binding to CA-125 were identified.

Philosophiae Doctor - PhD<br>Breast cancer is one of the major causes of death in South Africa. About 1 in 29 South African women are at risk of developing this type of cancer in their lifetime. The global incidence of breast cancer also increases annually with over 1 million new cases diagnosed every year. Molecular diagnostic techniques such as qRT-PCR, Fluorescent In Situ Hybridization (FISH), Immunohistochemistry (IHC) and ELISA are used to diagnose breast cancer. Some of these diagnostic techniques make use organic fluorophores as fluorescent reporter molecules. The principle of all these diagnostic techniques is reliant on the detection of molecular biomarkers that are associated with the disease. In most cases these molecular biomarkers are DNA, RNA or proteins that are up-regulated in response to or as a result of the disease. The first aim of this study was therefore to identify membrane proteins that are up-regulated in cancers that can potentially be used as biomarkers for the detection of breast cancer. The second aim of this study was to investigate the application of quantum dots in the development of a molecular diagnostic test that can detect a breast cancer biomarker. The most commonly used method to identify molecular biomarkers for diseases have traditionally been gene expression analysis using technologies such as DNA microarray. These technologies have certain limitations and have therefore not been very successful in identifying useful disease biomarkers. Biomarker II discovery by proteomics can overcome some of these limitations and is potentially a more suitable method to identify molecular biomarkers for breast cancer. In this study proteomics in combination with Stable Isotope Labelling with Amino Acids in Cell Culture SILAC was used to do a comparative analysis of the expression levels of membrane proteins present in a human breast cancer cell line (MCF-7) derived from a breast cancer patient and a human breast cell line (MCF- 12A) derived from a healthy individual. This led to the identification of the transmembrane protein, GFRA1 as potential new biomarker for breast cancer. This study showed that this protein is over expressed in MCF-7 cells as compared to MCF-12A cells and that it is also highly expressed in the myoepthelial cells of the milk ducts of breast cancer patients. This study also demonstrates the use of molecular beacon technology to develop a DNA probe for the detection of cDNA encoding the CK19 gene, which is a known biomarker for breast cancer. In the development of this probe, quantum dots were used as the fluorescence reporter. This molecular beacon probe was able to demonstrate the over expression of CK19 in MCF-7 cells. This study shows that this technology can potentially be used as a diagnostic test for breast cancer and since quantum dots are used in the development of these molecular beacon probes, this diagnostic test can potentially facilitate the development of multiplex detection systems for the diagnosis of breast cancer. Molecular beacon technology can potentially also be used to detect novel biomarkers such as GFRA1.

This thesis is about the design of a biosensor system for detection of multiple cancer biomarkers. Accurate diagnosis and prognosis of cancer requires early detection. Equally important, though, is the measurement of biomarker-velocity and detection of multiple biomarkers. Early detection requires highly sensitive biosensors capable of detection at very low concentrations of target molecules. Biomarker-velocity can be measured by monitoring concentration of target molecule over a period of time. This requires a system which is very easy to use, fast, flexible, inexpensive and portable, thus enabling its ubiquitous presence at the point of care. For detection of multiplexed biomarkers, biosensors which easily lend to array configuration are required. Conventional techniques do not fulfill either all or some aspects of the requirements listed above. In this work, we present the design of a biosensor system, keeping in view the desired features described above, to achieve the ultimate goal of enabling ubiquitous presence of biosensor at the point of care. We focus on acoustic transducer based biosensors. The two fundamental components of design in an acoustic biosensor are the design of an acoustic transducer and the design of a novel electrical interface for the transducer. For transducer design, we introduce and present the design of a single structure, GHz range, multi-mode acoustic resonator. We present this as a suitable transducer for liquid phase biosensors, which is the preferred medium for sensing of cancer biomarkers. We explore the underlying physics and do experimental and theoretical characterization of this device. The transducer needs to be functionalized with a chemically sensitive layer which performs the molecular recognition of cancer biomarkers. We present the experimental exploration of a reversible and oriented immobilization based Histidine-Ni(2+) interaction which used NTA as the chelator for anchoring onto the device. Then we discuss the microfluidic design to enable liquid phase operation. We used SU-8 polymer barriers for liquid containment and addressed the challenges of making it compatible with ZnO based devices. An electrical interface is needed to excite and extract the sensor response. We have presented here a novel method to measure and track a resonator's response and extract its characteristic parameters. This method measures the wideband frequency response of the resonator with a much simpler setup as compared to conventional methods. We have proposed and demonstrated the use of a white noise signal as a viable signal for broadband excitation of resonator-based sensing platforms. We have also established, shown through simulation and prototype measurements, the feasibility of the proposed method. The accuracy and speed of the system can be further greatly improved by FFT-based digital implementation of the spectral analysis system. We have presented an example hardware implementation of FFT-based signal analyzer, and have discussed the hardware resources required for actual implementation in a chip form. Lastly we discuss the measurement protocol and sensor results for head and neck cancer and prostate cancer biomarkers. These results demonstrate the usability of the proposed sensor system for detection of cancer biomarkers.

[ES] El cuidado de la salud es un campo en el que la detección precoz de enfermedades está cobrando cada vez más importancia. Hoy en día, profesionales y ciudadanos demandan que las técnicas de diagnóstico sean de alta calidad, tanto para el sistema de sanidad privado como para el público. Cuando se utilizan técnicas de diagnóstico de manera inadecuada, eso puede acarrear bastantes consecuencias, tales como un serio peligro sobre la salud y la sobrecarga técnica y económica de los servicios de salud. Eso es debido a que las técnicas de diagnóstico disponibles hoy en día son demasiado costosas, centralizadas en laboratorios y necesitan profesionales altamente cualificados para poder llevar a cabo dichas tareas, lo que conllevaría una demora en el tiempo, siendo este muchas veces vital para los enfermos. Es muy necesario, por lo tanto, reflexionar sobre la necesidad y emergencia de tales prácticas preventivas, especialmente para enfermedades de alto riesgo como el cáncer, el Alzheimer o la primera causa de muerte en el mundo, las enfermedades cardiovasculares. En este contexto, el objetivo principal del trabajo realizado durante esta Tesis Doctoral es ayudar a superar estos problemas mediante la exploración de la posibilidad de utilizar tecnología fotónica para el desarrollo de sistemas de análisis que puedan ser utilizados para el diagnóstico y pronóstico de las enfermedades cardiovasculares. Este objetivo se ha abordado mediante la combinación de la tecnología nanofotónica, consistiendo en la nanofabricación de las estructuras PBG de sensado que ofrece varios beneficios, como una alta sensibilidad, una extrema reducción de tamaño y un proceso de fabricación compatible con el de la industria microelectrónica, con un método de biofuncionalización obteniendo una capa de bioreconocimiento estable y selectiva mediante el uso de la reacción TEC asistida por luz capaz de proporcionar unas capas de bio-reconocimiento extremadamente finas con una inmovilización espacialmente selectiva.<br>[CAT] L'atenció a la salut és un camp en què la detecció precoç de malalties està cobrant cada vegada més importància. Hui en dia, professionals i ciutadans demanen que les tècniques de diagnòstic siguin d'alta qualitat, tant per al sistema de sanitat privat com per al públic. Quan s'utilitzen tècniques de diagnòstic de manera inadequada, això pot comportar bastants conseqüències, com ara, un seriós perill sobre la salut i la sobrecàrrega tècnica i econòmica dels serveis de salut. Això és degut al fet que les tècniques de diagnòstic disponibles hui en dia són molt costoses, centralitzades en laboratoris i necessiten professionals altament qualificats per poder realitzar aquestes tasques, lo que comportaria a una demora en el temps que moltes vegades es vital pels malalts. És molt necessari, per tant, reflexionar sobre la necessitat i emergència de tals practiques preventives, especialment per a malalties d'alt risc com el càncer, l'Alzheimer o la primera causa de mort al món, les malalties cardiovasculars. En aquest context, l'objectiu principal del treball realitzat durant aquesta Tesi Doctoral és ajudar a superar aquests problemes mitjançant l'exploració de la possibilitat d'utilitzar tecnologia fotònica per al desenvolupament de sistemes d'anàlisis que puguin ser utilitzats per al diagnòstic i pronòstic de les malalties cardiovasculars. Aquest objectiu s'ha abordat mitjançant la combinació de la tecnologia nanofotònica, consistint en la nanofabricació de les estructures de detecció de PBG fotòniques que ofereix diversos beneficis, com una alta sensibilitat, una extrema reducció de mida i un procés de fabricació compatible amb el de la indústria microelectrònica, amb un mètode de biofuncionalització obtenint una capa de bio-reconeixement estable i selectiva mitjançant l'ús de la reacció TEC assistida per llum capaç de proporcionar unes capes de bioreconeixement extremadament fines amb una immobilització espacialment selectiva. preventives, especialment per a malalties d'alt risc com el càncer, l'Alzheimer o la primera causa de mort al món, les malalties cardiovasculars. En aquest context, l'objectiu principal del treball realitzat durant aquesta Tesi Doctoral és ajudar a superar aquests problemes mitjançant l'exploració de la possibilitat d'utilitzar tecnologia fotònica per al desenvolupament de sistemes d'anàlisis que puguin ser utilitzats per al diagnòstic i pronòstic de les malalties cardiovasculars. Aquest objectiu s'ha abordat mitjançant la combinació de la tecnologia nanofotònica, consistint en la nanofabricació de les estructures de detecció de PBG fotòniques que ofereix diversos beneficis, com una alta sensibilitat, una extrema reducció de mida i un procés de fabricació compatible amb el de la indústria microelectrònica, amb un mètode de biofuncionalització obtenint una capa de bio-reconeixement estable i selectiva mitjançant l'ús de la reacció TEC assistida per llum capaç de proporcionar unes capes de bioreconeixement extremadament fines amb una immobilització espacialment selectiva.<br>[EN] Healthcare is a field where the early detection of diseases is becoming more and more important. Nowadays, professionals and citizens demand high quality diagnosis techniques offered by both private and public health systems. When the application of diagnostic tests is not adequate, different consequences can be observed such as health hazard and technical and economic overload of health services. This is due to the fact that the diagnostic techniques available are expensive, centralized in laboratories and with the need for highly qualified professionals to carry out these tasks, what can fundamentally lead to delays in time, being critical for the patient's health. It is very necessary, therefore, to reflect on the need and emergency of such preventive practices, especially for high-risk diseases such as cancer, Alzheimer or the first cause of death in the world, the cardiovascular diseases. Within this context, the main objective of the work done during this PhD Thesis is to help on overcoming these problems by exploring the possibility of using photonic technology for the development of analysis devices which might be used for the early diagnosis and prognosis of cardiovascular diseases. This objective has been addressed by combining nanophotonic technology, by the nanofabrication of the photonic PBG sensing structures, which provides several benefits such as a high sensitivity, an extreme size reduction and a fabrication process being compatible with that from the microelectronics industry, with a light-assisted biofunctionalization method forming a stable and selective biorecognition layer using TEC reaction able to provide extremely thin biorecognition layers with a spatially-selective immobilization.<br>Sabek, J. (2019). Combination of nanophotonic biosensors and light-assisted immobilization procedures for the detection of cardiac biomarkers [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/124821<br>TESIS