Surface-enhanced Raman spectroscopy for forensic analysis of human saliva

Through oral contact, saliva can be transferred to a variety of objects commonly encountered in forensic casework, such as cigarette butts, condoms, drinkware, masks, clothing, and vaginal or penile swabs. As saliva does not require external stimulation for secretion, it is readily available for exposure to the external environment. For this reason, the detection and analysis of saliva are relevant to many types of crime, including but not limited to sexual assaults, kidnappings, homicides, and burglaries. Although the presence of saliva does not serve as a definitive indication of crime, it can link individuals to a crime scene or serve as evidence of physical contact. When sufficient quality and quantity of saliva is present at a crime scene or on an item of evidence, it typically serves as a source of DNA.1 Therefore, detection of saliva is also pertinent to the identification of potential victims and perpetrators. Currently, forensic identification of biological stains is limited to individual body fluids, such that analysis of a mixture containing multiple body fluids will require a separate test for each biological component. In addition, all routine forensic assays for saliva are of a preliminary nature. Due to the lack of confirmation techniques for saliva, surface-enhanced Raman spectroscopy (SERS) emerges as a potential technique for rapid, sensitive, and confirmatory identification of saliva stains. This study is designed to examine the efficacy of a SERS based methodology for the detection, characterization, and identification of human saliva. Using this technology, the major molecular component detected in saliva was salivary thiocyanate. A three-peak thiocyanate SERS signature was observed at 440 cm-1, 740 cm-1, and 2107 cm-1 and is recommended for use as a biomarker in the detection and identification of liquid and dried saliva. Assessment of validation parameters was also performed to determine the robustness, accuracy, and sensitivity of this methodology. Examination of donor variability among and within individuals demonstrates variability in salivary thiocyanate concentrations. As a result, the sensitivity of this technology is limited by the inherent thiocyanate concentrations of individuals. In the samples utilized for this study, SERS could readily detect salivary thiocyanate at concentrations as low as 1/100. Classification of spectral data from 12 donors indicates some donor variability arising from differences in protein content. However, deviations in matrix proteins do not hinder the identification of saliva because this methodology uses a salivary thiocyanate SERS signature for the basis of saliva detection. A preliminary sample aging study demonstrates that saliva can be rapidly identified in 20-month old samples aged at room temperature. This suggests that the salivary thiocyanate SERS signature is stable without the need for controlled environments and, therefore, highly suitable for use in forensic detection of human body fluids. At many crime scenes, biological fluids are present in a mixed sample consisting of human fluids such as saliva, semen, blood, and vaginal fluid. Salivary mixtures with blood and vaginal fluid were respectively analyzed to assess the efficiency of this SERS methodology in detecting and identifying saliva from the mixture. As thiocyanate is also an inherent component of blood, the ability to identify saliva from a salivary mixture with blood is limited to mixtures where saliva is a predominant component. For samples used in this study, saliva identification is limited to mixtures containing 75% saliva and 25% blood. As vaginal fluid does not contain salivary thiocyanate, this SERS methodology can readily identify salivary thiocyanate from mixtures containing 1% saliva and 99% vaginal fluid extract. This study examined two potential substrates and their corresponding efficiencies in body fluid detection for forensic casework. The thiocyanate signature is readily observed on both gold (Au) and silver (Ag) substrates. With respect to sensitivity, Ag substrates are more sensitive and capable of detecting thiocyanate in lower concentrations of saliva. Au substrates exhibit greater selectivity for other salivary components at low thiocyanate concentrations, which can be useful in characterizing the protein content. In view of all experimental results, this study demonstrates that SERS is a rapid and sensitive technique capable of detecting and identifying saliva among complex body mixtures for forensic science applications

Modification of Gold Nanoparticles for SERS Application in Emulsion and Lipid Systems

Gold nanoparticles produced using the Turkevich method were able to have their hydrophobicity modified using octanethiol in a novel method for SERS application. Both amphiliphic GNPs and hydrophobic GNPs were produced and differentiated by Raman signals. The amphiliphic GNPs were able to enhance the SERS signals of the protein emulsifier in the emulsion in situ and the hydrophobic GNPs were able to enhance the SERS signals from canola oil. Further purification of the hydrophobic GNPs proved to have higher enhancement and sensitivity, but still poor consistency which is typical of SERS. Monitoring lipid oxidation using Raman and SERS using alternative approaches was the primary objective of the thesis. The purified GNPs were capable of enhancing the canola oil over two weeks, but the poor consistency led to no major trends. Using normal Raman, a triphenylphosphine oxidation reaction was capable of producing a peroxide value correlation in a very simple and rapid manor. Using a gold nanoparticle modified stainless steel wire, the headspace volatiles from canola oil oxidation were able to be enhanced, but with poor consistency. Silver dendrites were used to enhance the canola oil signal but with poor resolution. A combination of silver dendrites and GNPs were used to slightly improve enhancement, but not as strong as GNPs alone

Paper-based microfluidic devices for food adulterants: Cost-effective technological monitoring systems

Financiado para publicación en acceso aberto: Universidade de Vigo/CISUGAnalytical sciences have witnessed emergent techniques for efficient clinical and industrial food adulterants detection. In this review, the contributions made by the paper-based devices are highlighted for efficient and rapid detection of food adulterants and additives, which is the need of the hour and how different categories of techniques have been developed in the past decade for upgrading the performance for point-of-care testing. A simple strategy with an arrangement for detecting specific adulterants followed by the addition of samples to obtain well-defined qualitative or quantitative signals for confirming the presence of target species. The paperbased microfluidics-based technology advances and prospects for food adulterant detection are discussed given the high-demand from the food sectors and serve as a valued technology for food researchers working in interdisciplinary technological frontiers.Vision Group on Science and Technology, Government of Karnataka | Ref. KSTePS/ VGST/SMYSR-2016–17/GRD-595/2017–18Vision Group on Science and Technology, Government of Karnataka | Ref. KSTePS/VGSTRGS/F/GRD No.711/2017–18Science and Engineering Research Board (SERB), Department of Science and Technology, Govt of India | Ref. CRG/2020/00306

Probing the impact of sustainable emerging sonication and DBD plasma technologies on the quality of wheat sprouts juice

Financiado para publicación en acceso aberto: Universidade de Vigo/CISUGSonication and dielectric barrier discharge (DBD) plasma are sustainable emerging food processing technologies. The study investigates the impact of sonication, DBD-plasma, and thermal treatment (TT) on wheat sprout juice. The obtained results indicated a significant (p < 0.05) increase in chlorophyll, total phenolics, flavonoids, DPPH assay, and ORAC assay after DBD-plasma (40 V) and sonication (30 mins) treatment as compared to TT and untreated samples. Both emerging technologies significantly (p < 0.05) reduce the polyphenol oxidase and peroxidase activities, but the TT sample had the highest reduction. Moreover, the synergistic application of both technologies significantly reduced the E. coli/Coliform, aerobics, yeast and mold up to the 2 log reduction, but the TT sample had a complete reduction. DBD-plasma and sonication processing significantly decreased (p < 0.05) the particle size, reducing apparent viscosity (η) and consistency index (K); while increasing the flow behavior (n), leading to higher stability of wheat sprout juice. To assess the impact of emerging techniques on nutrient concentration, we used surface-enhance Raman spectroscopy (SERS) as an emerging method. Silver-coated gold nano-substrates were used to compare the nutritional concentration of wheat sprout juice treated with sonication, DBD-plasma, and TT-treated samples. Results showed sharp peaks for samples treated with DBD-plasma followed by sonication, untreated, and TT. The obtained results, improved quality of wheat sprout juice, and lower microbial and enzymatic loads were confirmed, showing the suitability of these sustainable processing techniques for food processing and further research

LoC-SERS technique toward drug quantification in complex materials

LoC-SERS Technique Toward Drug Quantification in Complex Matrices Kumulative Dissertation Zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) vorgelegt dem Rat der Chemisch-Geowissenschaftlichen Fakultät der Friedrich-Schiller-Universität Jena M. Sc. Izabella Jolán Hidi geboren am 27. Februar 1987 in Deva, Rumänien Abstract Detection and quantification of drugs in complex matrices challenged each analytical method at its moment of development and implementation. On one side, clinical samples, i.e. blood and urine, are routinely analyzed for diagnosis and treatment follow-up purposes. On the other side, monitoring the successful removal of pharmaceutically active compounds in waste and surface water is of high interest to overcome the occurrence of antibacterial resistance in the ecosystem. Among the newly developed analytical techniques, surface enhanced Raman spectroscopy (SERS) has high potential to be considered as an alternative analytical technique for the detection of clinically and environmentally relevant molecules in complex matrices. The biggest barrier for its application is the low reproducibility of measurements and automation. A solution that requires low-cost SERS substrates and offers reproducible and automated measurement conditions at the same time is represented by the droplet based lab-on-a-chip SERS (LoC-SERS) technique. This platform offers the advantage of highly precise controlled manipulation of nanoparticle and sample solution via computer controlled systems and a direct, on chip detection. The aim of the present thesis was to assess the potential and limitations of the LoC-SERS technique to detect and quantify drugs in various complex matrices. As SERS active substrates, easy-to-prepare silver nanoparticles in a colloidal solution were employed and as sample clean-up procedure filtration and dilution with water was carried out. Urine samples originating from healthy volunteers and patients with urinary tract infection, as well as river water sample were included in the present study. Analytical performances, such as lower limit of detection (LOD), linear and dynamic range and measurement reproducibility, were tested for methotrexate, levofloxacin, nitroxoline and ciprofloxacin. Furthermore, the potential of the LoC-SERS platform for on-site applications is also demonstrated by replacing the bench-top Raman microscope by a portable one

Ultra-Small Silver Nanoparticles - Dynamic Behavior in Aqueous and Biological Environments

The utilization of silver nanoparticles in consumer related products has significantly increased over the last decade, especially due to their antimicrobial properties. Today they are used in a plethora of products ranging from cosmetics and textiles to medical instruments. Thus, investigations on nanoscale silver are attracting increasing interest in many fields, such as biomedicine or catalysis. Unfortunately, the results of these studies are diverse and do not lead to a consistent evaluation of the toxicity of silver nanoparticles. A major flaw is the usage of non-uniform and inadequate characterized particles with broad size distributions. To elucidate this problem this thesis deals with the synthesis and thorough characterization of a versatile silver nanoparticle system in a size at the lower end of the nanoscale. Poly(acrylic acid) (PAA)-stabilized silver nanoparticles with a nominal radius of 3 nm are synthesized using two methods. Firstly, they are produced in a classical batch synthesis and secondly in a microwave-assisted synthesis. The comparison of these two routes applying the same reaction conditions provides the opportunity to reveal whether non-thermal microwave effects, which are still under debate in literature, are present. The synthesized particles exhibit a high stability and are used in an inter-laboratory comparison proving their suitability for application in nanometrology, such as determination of nanoparticle sizes, size distribution widths, and particle concentrations. Furthermore, the high versatility of the silver nanoparticle system enables an easy ligand exchange to tune the surface of the particles on demand and improve their biocompatibility. Exemplarily, transfunctionalizations with albumin, glutathione, and tyrosine are performed and characterized by small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), UV/Vis and IR spectroscopy. These differently coated silver nanoparticles are used firstly in a catalytic application: the reduction of 4-nitrophenol. Therein, a dependence of the catalytic activity on the corresponding coating is observed and reveals that the PAA-stabilized silver nanoparticles exhibit an exceptionally high activity. As a preliminary study for future biological applications the binding behavior, especially the adsorption and desorption, of biocompatible, fluorescent ligands is investigated. It is demonstrated that a fluorescent labelling with appropriate binding properties can be provided, which is subsequently used in an initial investigation on lung and intestinal cells regarding particle transport. Subsequently, the PAA-stabilized particles are employed in an artificial digestion. The monitoring of the size and size distribution throughout the three steps of digestion (saliva, stomach, intestine) shows that the silver nanoparticles pass the digestion under transformation in size, but still remain nano-sized. Since silver is known for the continuous release of silver ions, the digestion of silver nanoparticles leads naturally to the question of the behavior of ionic silver in an artificial digestion. The investigation of this topic with the help of small and wide-angle X-ray scattering and IR spectroscopy reveals that silver thiocyanate nanoparticles are formed during in vitro digestion. The release of silver ions from the surface of silver nanoparticles is a crucial point in the risk assessment of silver nanoparticles, since the ions can undergo complex transformations in biological environments. In this work, enhanced ion release was observed for the interaction of PAA-stabilized silver nanoparticles with glutathione. New small silver clusters were formed during this process. The diverse applications of the silver nanoparticle system in this work demonstrate the high versatility and stability of the system, which is a promising candidate for further comparable biological and catalytic applications.Die Verwendung von Silbernanopartikeln in Konsumgütern des täglichen Lebens hat in den letzten zehn Jahren deutlich zugenommen, insbesondere aufgrund ihrer antimikrobiellen Eigenschaften. Heute werden sie in einer Vielzahl von Produkten eingesetzt, die von Kosmetika und Textilien bis hin zu medizinischen Instrumenten reichen. So stoßen Untersuchungen an Nanosilber in vielen Bereichen wie der Biomedizin oder der Katalyse auf zunehmendes Interesse. Leider sind die Ergebnisse dieser Studien divers und führen nicht zu einer konsistenten Bewertung der Toxizität von Silbernanopartikeln. Eine große Schwachstelle ist die Verwendung von uneinheitlichen und unzureichend charakterisierten Partikeln mit breiten Größenverteilungen. Um dieses Problem zu beleuchten, beschäftigt sich diese Arbeit mit der Synthese und umfassenden Charakterisierung eines vielseitigen Silbernanopartikelsystems mit einer Größe am unteren Ende der Nanoskala. Poly(acrylsäure) (PAA)-stabilisierte Silbernanopartikel mit einem nominalen Radius von 3 nm werden mit zwei Methoden synthetisiert. Zum einen werden sie in einer klassischen Batch-Synthese und zum anderen in einer mikrowellengestützten Synthese hergestellt. Der Vergleich dieser beiden Verfahren bei gleichen Reaktionsbedingungen ermöglicht die Aufklärung, ob nicht-thermische Mikrowelleneffekte, die in der Literatur noch diskutiert werden, in diesem Fall vorhanden sind. Die synthetisierten Partikel weisen eine hohe Stabilität auf und werden in einem Ringversuch eingesetzt, um ihre Eignung für den Einsatz in der Nanometrologie, wie zum Beispiel der Bestimmung von Nanopartikelgrößen, Größenverteilungsbreiten und Partikel-konzentrationen, nachzuweisen. Darüber hinaus ermöglicht die hohe Vielseitigkeit des Silbernanopartikelsystems einen einfachen Ligandenaustausch, um die Oberfläche der Partikel nach Bedarf anzupassen und ihre Biokompatibilität zu verbessern. Beispielhaft werden Umfunktionalisierungen mit Albumin, Glutathion und Tyrosin durchgeführt und durch Röntgenkleinwinkelstreuung (SAXS), dynamische Lichtstreuung (DLS), UV/Vis- und IR-Spektroskopie charakterisiert. Diese unterschiedlich beschichteten Silbernanopartikel werden zunächst in einer katalytischen Anwendung eingesetzt: Der Reduktion von 4-Nitrophenol. Dabei wird die Abhängigkeit der katalytischen Aktivität von der entsprechenden Beschichtung beobachtet und es zeigt sich, dass die PAA-stabilisierten Silbernanopartikel eine außergewöhnlich hohe Aktivität aufweisen. Als Vorbetrachtung für zukünftige Anwendungen der Silbernanopartikel in biologischen Fragestellungen wird das Bindungsverhalten, insbesondere die Adsorption und Desorption, von biokompatiblen, fluoreszierenden Liganden untersucht. Es zeigt sich, dass eine Fluoreszenzmarkierung mit geeigneter Bindungswirkung bereitgestellt werden kann. Die fluoreszenzmarkierten Partikel werden anschließend in einer ersten Untersuchung hinsichtlich des Partikeltransports auf Lungen- und Darmzellen eingesetzt. Anschließend werden die PAA-stabilisierten Partikel in einem künstlichen Verdau untersucht. Die Verfolgung der Größe und Größenverteilung in den drei Verdaustufen (Speichel, Magen, Darm) zeigt, dass die Silbernanopartikel den Verdau unter Größenveränderungen passieren, aber dennoch nanoskalig bleiben. Da Silber für die kontinuierliche Freisetzung von Silberionen bekannt ist, führt der Verdau von Silbernanopartikeln zu der Frage nach dem Verhalten von ionischem Silber in einem künstlichen Verdau. Die Untersuchung dieses Themas mit Hilfe von Röntgenklein- und Röntgenweitwinkelstreuung, sowie IR-Spektroskopie zeigt, dass bei dem in vitro Verdau Silberthiocyanatnanopartikel entstehen. Die Freisetzung von Silberionen von der Oberfläche von Silbernanopartikeln ist ein entscheidender Punkt bei der Risikobewertung von Silbernanopartikeln, da die Ionen in biologischen Umgebungen komplexe Transformationen durchlaufen können. In dieser Arbeit wurde eine erhöhte Ionenfreisetzung für die Interaktion von PAA-stabilisierten Silbernanopartikeln mit Glutathion beobachtet. Dabei entstanden neue kleine Silbercluster. Die vielfältigen Anwendungen des Silbernanopartikelsystems in dieser Arbeit zeigen die hohe Vielseitigkeit und Stabilität des Systems, das ein vielversprechender Kandidat für weitere vergleichbare biologische und katalytische Anwendungen ist

NanoSERS Microfluidics platform for rapid screening for infectious diseases

Early and accurate disease detection is critical for clinical diagnosis and ultimately determining patient outcomes. Point-of-care testing (POCT) platforms are needed in low- resource settings and also to help the decentralisation of healthcare centres. Immunoas- says using Surface-Enhanced Raman Spectroscopy (SERS) are especially interesting for their increased sensitivity and specificity. Additionally, SERS can be easily translated into POCT formats with microfluidics. In this work, a sensitive, selective, capable of multiplexing, and reusable SERS-based biosensor was developed. The SERS immunoas- say relies on a sandwich format, whereby a capture platform and SERS immunotags can capture and detect a specific antigen, respectively. The SERS immunotags consisted of gold nanostars, allowing exceptionally intense SERS signals from attached Raman re- porters, and the covalent attachment of antibodies provided a stable antigen-antibody binding activity. As a capture platform, a regenerated cellulose-based hydrogel provided a robust design and the added advantage of environmental friendliness. Besides being a transparent material with low background fluorescence and Raman signal, its high-water retention capacity was particularly suited for preserving the high activity of covalently bound antibodies, improving the assay time-stability. This SERS-based immunoassay was then integrated into a microfluidic device, allowing high-throughput sample screening allied with the high sensitivity and multiplexing features of the developed assay. The de- vice was fabricated in less than 30 minutes by exploring direct patterning on shrinkable polystyrene sheets for the construction of adaptable complex three-dimensional microflu- idic chips. Finally, to validate the microfluidic system, Plasmodium falciparum infected red blood cell culture samples were tested for malaria biomarker detection. The discrimi- nation of SERS immunotags signals from the background was made through the direct classical least squares method. As a result, better data fitting was achieved, compared to the commonly used peak integral method. Considering these features, the proposed SERS-based immunoassay notably improved the detection limits of traditional enzyme- linked immunosorbent assay approaches. Its performance was better or comparable to existing SERS-based immunosensors. Moreover, this approach successfully overcame the main challenges for application at POCT, including increasing reproducibility, sensitivity, and specificity. Hence, the microfluidic SERS system represents a powerful technology which can contribute to early diagnosis of infectious diseases, a decisive step towards lowering their still substantial burden on health systems worldwide.A detecção precoce e precisa de doenças é fundamental para o diagnóstico clínico de- terminando frequentemente o prognóstico do paciente. Desta forma, plataformas de teste de rastreio (conhecidos pelo acrónimo de POCT) são extremamente necessárias, não só em locais com poucos recursos, mas também para ajudar à descentralização dos cuidados de saúde. Os ensaios imunológicos que utilizam a espectroscopia de Raman aumentada pela superfície (conhecida pelo acrónimo de SERS) são particularmente interessantes pela sua elevada sensibilidade. Além disso, os ensaios em SERS podem ser facilmente convertidos para formatos POCT quando combinados com microfluídica. Este trabalho consistiu no desenvolvimento de um biosensor sensível, selectivo, capaz de múltipla detecção e reuti- lizável baseado no fenómeno de SERS. O ensaio imunológico em SERS foi realizado num formato em sanduíche onde um antigénio específico é apreendido por uma plataforma de captura e reconhecido por imunosondas activas em SERS. Estas sondas consistem em nanopartículas de ouro em forma de estrela, que proporcionam um sinal de SERS intenso proveniente das moléculas repórter de Raman ligadas às nanopartículas. As sondas ad- quirem a especificidade necessária para o antigénio de anticorpos a elas ligados de forma covalente, e, por conseguinte, permitem uma ligação estável antigénio-anticorpo. O hidro- gel regenerado à base de celulose forneceu uma plataforma de captura de design robusto e ecológico. Além de ser um material transparente com baixa fluorescência e, portanto, de baixa interferência no sinal de Raman, é um material com uma elevada capacidade de retenção de água tornando-o particularmente adequado para preservar a actividade dos anticorpos ligados covalentemente. Deste modo, o hidrogel proporciona uma plataforma de captura estável ao longo do tempo. O immunoensaio baseado em SERS desenvolvido foi posteriormente integrado num dispositivo de microfluídica, permitindo analisar um grande número de amostras sendo simultaneamente sensível e passível para aplicações de análise de múltiplos antigénios. O dispositivo foi fabricado em menos de 30 minu- tos devido à padronização directa em folhas de poliestireno contrácteis possibilitando a construção tridimensional de um dispositivo de microfluídica. Finalmente, para validar o sistema de microfluídica, amostras de cultura de eritrócitos infectados com Plasmodium falciparum foram testadas para detecção de biomarcadores de malária. A discriminação dos sinais das immunosondas activas em SERS, relativamente a sinais interferentes, foi feita através do método clássico de quadrados mínimos. Como resultado, foi conseguido um melhor ajuste de dados em comparação com o método de cálculo do integral das áreas das bandas habitualmente utilizado. Assim, o ensaio imunológico baseado em SERS proposto neste trabalho permitiu obter um limite de detecção mais baixo do que o obtido pelas abordagens tradicionais como o ensaio de imunoabsorção enzimática (conhecido pelo acrónimo de ELISA), além de exibir um desempenho melhor ou comparável a ou- tros sensores baseados em SERS já existentes na literatura. Adicionalmente, o sistema desenvolvido neste trabalho permite ultrapassar desafios que impedem a utilização deste tipo de sensores em locais de poucos recursos, apresentando valores elevados de repro- dutibilidade, sensibilidade e especificidade. Por conseguinte, um sistema que combina SERS e microfluídica representa uma tecnologia potencialmente importante na detecção precoce, na esperança de que, num futuro próximo, as consequências das doenças infecci- osas que ainda impõem um fardo substancial ao sistema de saúde a nível mundial, sejam minoradas

Cancer theranostics: multifunctional gold nanoparticles for diagnostics and therapy

Doctorate in Biology, Specialty in BiotechnologyThe use of gold nanoparticles (AuNPs) has been gaining momentum in molecular diagnostics due to their unique physico-chemical properties these systems present huge advantages, such as increased sensitivity, reduced cost and potential for single-molecule characterisation. Because of their versatility and easy of functionalisation, multifunctional AuNPs have also been proposed as optimal delivery systems for therapy (nanovectors). Being able to produce such systems would mean the dawn of a new age in theranostics (diagnostics and therapy)driven by nanotechnology vehicles. Nanotechnology can be exploit for cancer theranostics via the development of diagnostics systems such as colorimetric and imunoassays, and in therapy approaches through gene therapy, drug delivery and tumour targeting systems. The unique characteristics of nanoparticles in the nanometre range, such as high surface-tovolume ratio or shape/size-dependent optical properties, are drastically different from those of their bulk materials and hold pledge in the clinical field for disease therapeutics This PhD project intends to optimise a gold-nanoparticle based technique for the detection of oncogenes’ transcripts (c-Myc and BCR-ABL) that can be used for the evaluation of the expression profile in cancer cells, while simultaneously developing an innovative platform of multifunctional gold nanoparticles (tumour markers, cell penetrating peptides, fluorescent dyes) loaded with siRNA capable of silencing the selected proto-oncogenes, which can be used to evaluate the level of expression and determine the efficiency of silencing. This work is a part of an ongoing collaboration between Research Centre for Human Molecular Genetics, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Portugal and Biofunctional Nanoparticles and Surfaces Group, Instituto de Nanociencia de Aragón, Spain within a European project [NanoScieE+ - NANOTRUCK]. In order to achieve this goal we developed effective conjugation strategies to combine, in a highly controlled way, biomolecules to the surface of AuNPs with specific functions such as: ssDNA oligos to detect specific sequences and for mRNA quantification; Biofunctional spacers: Poly(ethylene glycol) (PEG) spacers used to increase solubility and biocompatibility and confer chemical functionality; Cell penetrating peptides: to overcome the lipophilic barrier of the cellular membranes and deliver molecules into cells using TAT peptide to achieve cytoplasm and nucleus; Quaternary ammonium: to introduce stable positively charged in gold nanoparticles surface; and RNA interference: siRNA complementary to a master regulator gene, the proto-oncogene c-Myc, that is implicated in cell growth, proliferation, loss of differentiation, and cell death. In order to establish that they are viable alternatives to the available methods, these innovative nanoparticles were extensively characterized on their chemical functionalization, ease of uptake, cellular toxicity and inflammation, and knockdown of MYC protein expression in several cancer cell lines and in in vivo models.Fundação para a Ciência e Tecnologia - (SFRH/BD/62957/2009); PTDC/BIO/66514/2006; NANOLIGHT-PTDC/QUI-QUI/112597/2009; Silencing the silencers via multifunctional gold nanoconjugates towards cancer therapy - PTDC/BBB-NAN/1812/201