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Liquid biopsies in myeloid malignancies, Abdulmawjood, {Bilal R. }, Roma-Rodrigues Catarina, Fernandes {Alexandra R. }, and Baptista {Pedro V. } , Cancer Drug Resistance, Volume 2, Number 4, p.1044–1061, (2019) Abstract

Hematologic malignancies are the most common type of cancer affecting children and young adults, and encompass diseases, such as leukemia, lymphoma, and myeloma, all of which impact blood associated tissues such as the bone marrow, lymphatic system, and blood cells. Clinical diagnostics of these malignancies relies heavily on the use of bone marrow samples, which is painful, debilitating, and not free from risks for leukemia patients. Liquid biopsies are based on minimally invasive assessment of markers in the blood (and other fluids) and have the potential to improve the efficacy of diagnostic/therapeutic strategies in leukemia patients, providing a useful tool for the real time molecular profiling of patients. The most promising noninvasive biomarkers are circulating tumor cells, circulating tumor DNA, microRNAs, and exosomes. Herein, we discuss the role of assessing these circulating biomarkers for the understanding of tumor progression and metastasis, tumor progression dynamics through treatment and for follow-up.

Tackling Imatinib Resistance via Au-nanoconjugates using A Cml Resistant Cell Line, Abdulmawjood, Bilal, Roma-Rodrigues Catarina, Baptista {Pedro V. }, and Fernandes {Alexandra R. } , Particle and Particle Systems Characterization, Volume 41, Number 1, (2024) Abstract

Chronic myeloid leukemia (CML) is a rare malignant proliferative hematopoietic disease due to overexpression of a tyrosine kinase (TK) derived from the breakpoint cluster region (BCR)-abelson tyrosine-protein kinase 1 (ABL1) gene fusion. Imatinib (IM), blocks this tyrosine kinase, and is the first line TK inhibitor (TKI) used in CML treatment. In a high percentage of CML patients, a poor response with relapse and disease progression is associated to acquisition of resistance through different mechanisms, including dysregulation of c-MYC proto-oncogene. Gold nanoparticles (AuNPs) are shown to allow improved efficacy in gene silencing approaches toward cancer therapy. Herein, the silencing potential of AuNPs functionalized with antisense oligonucleotides selectively targeting the e14a2 BCR-ABL1 or the c-MYC, alone and combination is evaluated. It is demonstrated efficient silencing of gene expression that translated to a downregulation of protein levels in IM resistant CML cells (K562-IM). This combination allowed for increased death of the malignant cells. These Au-nanoconjugates may be useful to tackle IM-resistance mechanisms, providing an additional tool for future combinatory schemes to fight CML with imatinib resistance.

Genetic biomarkers in chronic myeloid leukemia: What have we learned so far?, Abdulmawjood, Bilal, Costa Beatriz, Roma-Rodrigues Catarina, Baptista {Pedro V. }, and Fernandes {Alexandra R. } , International Journal of Molecular Sciences, nov, Volume 22, Number 22, (2021) Abstract

Chronic Myeloid Leukemia (CML) is a rare malignant proliferative disease of the hematopoietic system, whose molecular hallmark is the Philadelphia chromosome (Ph). The Ph chromosome originates an aberrant fusion gene with abnormal kinase activity, leading to the buildup of reactive oxygen species and genetic instability of relevance in disease progression. Several genetic abnormalities have been correlated with CML in the blast phase, including chromosomal aberrations and common altered genes. Some of these genes are involved in the regulation of cell apoptosis and proliferation, such as the epidermal growth factor receptor (EGFR), tumor protein p53 (TP53), or Schmidt-Ruppin A-2 proto-oncogene (SRC); cell adhesion, e.g., catenin beta 1(CTNNB1); or genes associated to TGF-β, such as SKI like proto-oncogene (SKIL), transforming growth factor beta 1 (TGFB1) or transforming growth factor beta 2 (TGFB2); and TNF-α pathways, such as Tumor necrosis factor (TNFA) or Nuclear factor kappa B subunit 1 (NFKB1). The involvement of miRNAs in CML is also gaining momentum, where dysregulation of some critical miRNAs, such as miRNA-451 and miRNA-21, which have been associated to the molecular modulation of pathogenesis, progression of disease states, and response to therapeutics. In this review, the most relevant genomic alterations found in CML will be addressed.

Molecular Beacon for Detection miRNA-21 as a Biomarker of Lung Cancer, Alexandre, Daniela, Teixeira Bernardo, Rico André, Valente Salete, Craveiro Ana, Baptista {Pedro V. }, and Cruz Carla , International Journal of Molecular Sciences, mar, Volume 23, Number 6, (2022) Abstract

Lung cancer (LC) is the leading cause of cancer-related death worldwide. Although the diagnosis and treatment of non-small cell lung cancer (NSCLC), which accounts for approximately 80% of LC cases, have greatly improved in the past decade, there is still an urgent need to find more sensitive and specific screening methods. Recently, new molecular biomarkers are emerging as potential non-invasive diagnostic agents to screen NSCLC, including multiple microRNAs (miRNAs) that show an unusual expression profile. Moreover, peripheral blood mononuclear cells’ (PBMCs) miRNA profile could be linked with NSCLC and used for diagnosis. We developed a molecular beacon (MB)-based miRNA detection strategy for NSCLC. Following PBMCs isolation and screening of the expression profile of a panel of miRNA by RT-qPCR, we designed a MB targeting of up-regulated miR-21-5p. This MB 21-5p was characterized by FRET-melting, CD, NMR and native PAGE, allowing the optimization of an in-situ approach involving miR-21-5p detection in PBMCs via MB. Data show the developed MB approach potential for miR-21-5p detection in PBMCs from clinical samples towards NSCLC.

Evaluation of miR-155 silencing using a molecular beacon in human lung adenocarcinoma cell line, Alexandre, Daniela, Fernandes {Alexandra R. }, Baptista {Pedro V. }, and Cruz Carla , Talanta, jul, Volume 274, (2024) Abstract

Lung cancer (LC) is a leading cause of global cancer-related deaths, highlighting the development of innovative methods for biomarker detection improving the early diagnostics. microRNAs (miRs) alterations are known to be involved in the initiation and progression of human cancers and can act as biomarkers for diagnostics and treatment. Herein, we develop the application of molecular beacon (MB) technology to monitor miR-155-3p expression in human lung adenocarcinoma A549 cells without complementary DNA synthesis, amplification, or expensive reagents. Furthermore, we produced gold nanoparticles (AuNPs) for delivering antisense oligonucleotides into A549 cells to reduce miR-155-3p expression, which was subsequently detectable using the MB. The MB was designed and structural characterized by Förster Resonance Energy Transfer (FRET)-melting, Circular Dichroism (CD), Nuclear magnetic resonance (NMR), and fluorometric experiments, and then the hybridization conditions were optimized for an in vitro approach involving the detection of miR-155-3p in total RNA extracted from A549 cell line. The expression profile of miR-155-3p was obtained by RT-qPCR. The results demonstrated that MB was properly designed and showed efficacy in targeting miR-155-3p. Furthermore, a limit of detection down to nanomolar concentration was achieved and the specificity of the biosensor was proved. Moreover, the self-assembly of ASOs with AuNPs exhibited exceptional target specificity, effectively silencing miR-155-3p. Notably, compared to lipid-based transfection agent, AuNPs displayed superior silencing efficiency. We highlighted the ability of MB to detect changes in the target gene expression after gene silencing. Overall, this innovative approach represents a promising tool for detecting various biomarkers at the same time, with potential applications in clinical settings.

Structural characterization and biological properties of silver(I) tris(pyrazolyl)methane sulfonate, Almeida, J., Roma-Rodrigues Catarina, Mahmoud {Abdallah G. }, {Guedes da Silva} Fátima {M. C. }, Pombeiro {Armando J. L. }, Martins {Luísa M. D. R. S. }, Baptista {Pedro V. }, and Fernandes {Alexandra R. } , Journal of Inorganic Biochemistry, oct, Volume 199, (2019) Abstract

The water-soluble 1D helical coordination polymer [Ag(Tpms)]n (1) [Tpms = tris(pyrazolyl)methane sulfonate, −O3SC(pz)3; pz = pyrazolyl] was synthesized and fully characterized, its single-crystal X-ray diffraction analysis revealing the ligand acting as a bridging chelate N3-donor ligand. The antiproliferative potential of 1 was performed on two human tumour cell lines, A2780 and HCT116, and in normal fibroblasts, with a much higher effect in the former cell line (IC50 of 0.04 μM) as compared to the latter cell line and to normal fibroblasts. Compound 1 does not alter cell cycle progression but interferes with the adherence of A2780 cells triggering cell apoptosis. Apoptosis appears to occur via the extrinsic pathway (no changes in mitochondria membrane potential, reactive oxygen species (ROS) and pro-apoptotic (B-cell lymphoma 2 (BCL-2) associated protein (BAX))/anti-apoptotic (BCL-2) ratio) being this hypothesis also supported by the presence of silver mainly in the supernatants of A2780 cells. Results also indicated that cell death via autophagy was triggered. Proteomic analysis allowed us to confirm that compound 1 is able to induce a stress response in A2780 cells that is related with its antiproliferative activity and the trigger of apoptosis.

Multifunctional microfluidic chip for optical nanoprobe based RNA detection - Application to Chronic Myeloid Leukemia, Alves, {Pedro Urbano}, Vinhas Raquel, Fernandes {Alexandra R. }, Birol {Semra Zuhal}, Trabzon Levent, Bernacka-Wojcik Iwona, Igreja Rui, Lopes Paulo, Baptista {Pedro Viana}, Águas Hugo, Fortunato Elvira, and Martins Rodrigo , Scientific Reports, dec, Volume 8, Number 1, (2018) Abstract

Many diseases have their treatment options narrowed and end up being fatal if detected during later stages. As a consequence, point-of-care devices have an increasing importance for routine screening applications in the health sector due to their portability, fast analyses and decreased cost. For that purpose, a multifunctional chip was developed and tested using gold nanoprobes to perform RNA optical detection inside a microfluidic chip without the need of molecular amplification steps. As a proof-of-concept, this device was used for the rapid detection of chronic myeloid leukemia, a hemato-oncological disease that would benefit from early stage diagnostics and screening tests. The chip passively mixed target RNA from samples, gold nanoprobes and saline solution to infer a result from their final colorimetric properties. An optical fiber network was used to evaluate its transmitted spectra inside the chip. Trials provided accurate output results within 3 min, yielding signal-to-noise ratios up to 9 dB. When compared to actual state-of-art screening techniques of chronic myeloid leukemia, these results were, at microscale, at least 10 times faster than the reported detection methods for chronic myeloid leukemia. Concerning point-of-care applications, this work paves the way for other new and more complex versions of optical based genosensors.

Light Triggered Enhancement of Antibiotic Efficacy in Biofilm Elimination Mediated by Gold-Silver Alloy Nanoparticles, Alves-Barroco, Cinthia, Rivas-García Lorenzo, Fernandes {Alexandra R. }, and Baptista {Pedro Viana} , Frontiers in Microbiology, feb, Volume 13, p.1–15, (2022) Abstract

Bacterial biofilm is a tri-dimensional complex community of cells at different metabolic stages involved in a matrix of self-produced extracellular polymeric substances. Biofilm formation is part of a defense mechanism that allows the bacteria to survive in hostile environments, such as increasing resistance or tolerance to antimicrobial agents, causing persistent infections hard to treat and impair disease eradication. One such example is bovine mastitis associated with Streptococcus dysgalactiae subsp. dysgalactiae (SDSD), whose worldwide health and economic impact is on the surge. As such, non-conventional nanobased approaches have been proposed as an alternative to tackle biofilm formation and to which pathogenic bacteria fail to adapt. Among these, metallic nanoparticles have gained significant attention, particularly gold and silver nanoparticles, due to their ease of synthesis and impact against microorganism growth. This study provides a proof-of-concept investigation into the use of gold-silver alloy nanoparticles (AuAgNPs) toward eradication of bacterial biofilms. Upon visible light irradiation of AuAgNPs there was considerable disturbance of the biofilms' matrix. The hindering of structural integrity of the biofilm matrix resulted in an increased permeability for entry of antibiotics, which then cause the eradication of biofilm and inhibit subsequent biofilm formation. Additionally, our results that AuAgNPs inhibited the formation of SDSD biofilms via distinct stress pathways that lead to the downregulation of two genes critical for biofilm production, namely, brpA-like encoding biofilm regulatory protein and fbpA fibronectin-binding protein A. This study provides useful information to assist the development of nanoparticle-based strategies for the active treatment of biofilm-related infections triggered by photoirradiation in the visible.

Tackling Multidrug Resistance in Streptococci: From Novel Biotherapeutic Strategies to Nanomedicines, Alves-Barroco, Cinthia, Rivas-García Lorenzo, Fernandes {Alexandra R. }, and Baptista {Pedro Viana} , Frontiers in Microbiology, oct, Volume 11, (2020) Abstract

The pyogenic streptococci group includes pathogenic species for humans and other animals and has been associated with enduring morbidity and high mortality. The main reason for the treatment failure of streptococcal infections is the increased resistance to antibiotics. In recent years, infectious diseases caused by pyogenic streptococci resistant to multiple antibiotics have been raising with a significant impact to public health and veterinary industry. The rise of antibiotic-resistant streptococci has been associated to diverse mechanisms, such as efflux pumps and modifications of the antimicrobial target. Among streptococci, antibiotic resistance emerges from previously sensitive populations as result of horizontal gene transfer or chromosomal point mutations due to excessive use of antimicrobials. Streptococci strains are also recognized as biofilm producers. The increased resistance of biofilms to antibiotics among streptococci promote persistent infection, which comprise circa 80% of microbial infections in humans. Therefore, to overcome drug resistance, new strategies, including new antibacterial and antibiofilm agents, have been studied. Interestingly, the use of systems based on nanoparticles have been applied to tackle infection and reduce the emergence of drug resistance. Herein, we present a synopsis of mechanisms associated to drug resistance in (pyogenic) streptococci and discuss some innovative strategies as alternative to conventional antibiotics, such as bacteriocins, bacteriophage, and phage lysins, and metal nanoparticles. We shall provide focused discussion on the advantages and limitations of agents considering application, efficacy and safety in the context of impact to the host and evolution of bacterial resistance.

Light Irradiation of Gold Nanoparticles Toward Advanced Cancer Therapeutics, Amendoeira, Ana, García {Lorenzo Rivas}, Fernandes {Alexandra R. }, and Baptista {Pedro V. } , Advanced Therapeutics, jan, Volume 3, Number 1, (2020) Abstract

Cancer is one of the leading causes of death in the world. To challenge this epidemic, there are growing demands for the development of new advanced and targeted therapeutics capable of effectively tackling cancer cells with improved selectivity. Nanomedicine has put forward several innovative therapeutics toward improving therapeutic efficacy while decreasing the deleterious side effects of current chemotherapy. Multifunctional gold nanoparticles (AuNPs) have been at the core of a plethora of advanced therapeutic strategies that provide selective targeting with their unique optical properties, capable to interact with the light of specific wavelength to deliver therapy with tremendous spatiotemporal precision. AuNPs have been exploited as photodynamic and photothermal therapeutic agents alone or in combination with other cancer treatment modalities with other cancer applications. Due to their exceptional physicochemical properties, they have been proven efficacious allies for photodynamic therapy and for photothermal therapy regimens. Herein, the rapidly progressing literature related to the use of these promising strategies against cancer is discussed, highlighting their possible future clinical translation.

Cell Uptake of Steroid-BODIPY Conjugates and Their Internalization Mechanisms: Cancer Theranostic Dyes, Amendoeira, {Ana F. }, Luz André, Valente Ruben, Roma-Rodrigues Catarina, Ali Hasrat, {van Lier} {Johan E. }, Marques Fernanda, Baptista {Pedro V. }, and Fernandes {Alexandra R. } , International Journal of Molecular Sciences, feb, Volume 24, Number 4, (2023) Abstract

Estradiol-BODIPY linked via an 8-carbon spacer chain and 19-nortestosterone- and testosterone-BODIPY linked via an ethynyl spacer group were evaluated for cell uptake in the breast cancer cell lines MCF-7 and MDA-MB-231 and prostate cancer cell lines PC-3 and LNCaP, as well as in normal dermal fibroblasts, using fluorescence microscopy. The highest level of internalization was observed with 11β-OMe-estradiol-BODIPY 2 and 7α-Me-19-nortestosterone-BODIPY 4 towards cells expressing their specific receptors. Blocking experiments showed changes in non-specific cell uptake in the cancer and normal cells, which likely reflect differences in the lipophilicity of the conjugates. The internalization of the conjugates was shown to be an energy-dependent process that is likely mediated by clathrin- and caveolae-endocytosis. Studies using 2D co-cultures of cancer cells and normal fibroblasts showed that the conjugates are more selective towards cancer cells. Cell viability assays showed that the conjugates are non-toxic for cancer and/or normal cells. Visible light irradiation of cells incubated with estradiol-BODIPYs 1 and 2 and 7α-Me-19-nortestosterone-BODIPY 4 induced cell death, suggesting their potential for use as PDT agents.

Size-Dependent Biological Activities of Fluorescent Organosilane-Modified Zinc Oxide Nanoparticles, s}il{\u a}, Mariana Bu{\c, a}b{\u a}caru Aurel T. {\u, s}sat Viorica Mu{\c, Vasile {Bogdan S}tefan} {\c, Nea{\c s}u {Ionela Andreea}, Pinheiro Teresa, Roma-Rodrigues Catarina, Baptista {Pedro V. }, Fernandes {Alexandra R. }, Matos {António Pedro}, and Marques {Fernanda Marujo} , Journal of biomedical nanotechnology, feb, Volume 16, Number 2, p.137–152, (2020) Abstract

Surface modification of zinc oxide nanoparticles (ZnO NPs) is a strategy to tune their biocompatibility. Herein we report on the synthesis of a series of fluorescent ZnO NPs modified with 2-10% (3-glycidyloxypropyl)trimethoxysilane (GPTMS) to investigate the fluorescence properties and to explore their applications in microbiology and biomedicine. The obtained ZnO NPs were characterized by X-ray diffraction (XRD), high resolution transmission electron microscopy (HRTEM) and Fourier transform infrared spectroscopy (FTIR). Size reduction occurred from ca. 13 nm in unmodified ZnO to 3-4 nm in silane-modified samples and fluorescence spectra showed size-dependent variation of the photoemission bands' intensity. The antibacterial and cytotoxic activities were investigated on Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria, and in ovarian (A2780) and prostate (PC3) cancer cells by tetrazolium/formazan-based methods. The antibacterial effect was higher for E. coli than S. aureus, while the cytotoxic activity was similar for both cancer cells and varied with the particle size. Cell death by apoptosis, and/or necrosis versus autophagy, were explored by flow cytometry using an Annexin V based-method and transmission electron microscopy (TEM). The main mechanism of ZnO NPs toxicity may involve the generation of reactive oxygen species (ROS) and the induction of apoptosis or autophagy. This work revealed the potential utility of GPTMS-modified ZnO NPs in the treatment of bacterial infection and cancer.

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A promising road with challenges: where are gold nanoparticles in translational research?, Bao, Chenchen, Conde João, Polo Ester, {del Pino} Pablo, Moros Maria, Baptista Pedro, Grazu Valeria, Cui Daxiang, and {de la Fuente} {Jesus M. } , Nanomedicine, Volume 9, Number 15, p.2353–2370, (2014) Abstract

Nanoenabled technology holds great potential for health issues and biological research. Among the numerous inorganic nanoparticles that are available today, gold nanoparticles are fully developed as therapeutic and diagnostic agents both in vitro and in vivo due to their physicochemical properties. Owing to this, substantial work has been conducted in terms of developing biosensors for noninvasive and targeted tumor diagnosis and treatment. Some studies have even expanded into clinical trials. This article focuses on the fundamentals and synthesis of gold nanoparticles, as well as the latest, most promising applications in cancer research, such as molecular diagnostics, immunosensors, surface-enhanced Raman spectroscopy and bioimaging. Challenges to their further translational development are also discussed.

Gold and silver nanoparticles for clinical diagnostics - From genomics to proteomics., Baptista, {Pedro Miguel Ribeiro Viana} , Journal of Proteomics, jan, Volume 75, Number NA, p.2811–23, (2011) Abstract

Nanotechnology has prompted researchers to develop new and improved materials aimed at biomedical applications with particular emphasis in diagnostics and therapy. Special interest has been directed at providing enhanced biomolecular diagnostics, including SNP detection gene expression profiles and biomarker characterisation. These strategies have focused on the development of nanoscale devices and platforms that can be used for single molecule characterisation of nucleic acid, DNA or RNA, and protein at an increased rate when compared to traditional techniques. Also, several advances have been reported on DNA analysis in real time, at both high resolution and very high throughputs, suitable for biomedical diagnostics. Here, we shall provide a review of available nanotechnology-based platforms for biomolecular recognition, and their application to molecular diagnostics and genome analysis, with emphasis on the use of noble metal nanoparticles for simple and specific analysis systems. Particular focus will be put on those already being translated into clinical settings. This article is part of a Special Issue entitled: Clinical Proteomics.

Could gold nanoprobes be an important tool in cancer diagnostics?, Baptista, {Pedro Miguel Ribeiro Viana} , Expert Review Of Molecular Diagnostics, jan, Volume 12, Number 6, p.541–3, (2012) Abstract
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Gold nanoparticles for the development of clinical diagnosis methods, Baptista, Pedro, Pereira Eulália, Eaton Peter, c}alo Doria Gon{\c, Miranda Adelaide, Gomes Inês, Quaresma Pedro, and Franco Ricardo , Analytical and Bioanalytical Chemistry, jun, Volume 391, Number 3, p.943–950, (2008) Abstract

The impact of advances in nanotechnology is particularly relevant in biodiagnostics, where nanoparticle-based assays have been developed for specific detection of bioanalytes of clinical interest. Gold nanoparticles show easily tuned physical properties, including unique optical properties, robustness, and high surface areas, making them ideal candidates for developing biomarker platforms. Modulation of these physicochemical properties can be easily achieved by adequate synthetic strategies and give gold nanoparticles advantages over conventional detection methods currently used in clinical diagnostics. The surface of gold nanoparticles can be tailored by ligand functionalization to selectively bind biomarkers. Thiol-linking of DNA and chemical functionalization of gold nanoparticles for specific protein/antibody binding are the most common approaches. Simple and inexpensive methods based on these bio-nanoprobes were initially applied for detection of specific DNA sequences and are presently being expanded to clinical diagnosis.

Nanodiagnostics: Leaving the research lab to enter the clinics?, Baptista, {Pedro Viana} , Diagnosis, dec, Volume 1, Number 4, p.305–309, (2014) Abstract

Nanotechnology has provided a plethora of valuable tools that can be applied for the detection of biomolecules and analytes relevant for diagnosis purposes - nanodiagnostics. This surging new field of molecular diagnostics has been revolutionizing laboratory procedures and providing new ways to assess disease biomarkers with increased sensitivity. While most of the reported nanodiagnostics systems are proof-of-concepts that demonstrate their efficacy in the lab, several nanodiagnostics platforms have already matured to a level that open the way for effective translation to the clinics. Nanodiagnostics platforms (e.g., gold nanoparticles containing systems) have been remarkably useful for the development of molecular diagnosis strategies for DNA/RNA detection and characterization, including systems suitable for point-of-care. How near are nanodiagnostics to go from the bench to the bedside?

3h Gold nanobeacons: a potential nanotheranostics platform, Baptista, {Pedro Miguel Ribeiro Viana} , Nanomedicine, Volume 9, Number 15, p.2247–2250, (2014) Abstract
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Colorimetric detection of eukaryotic gene expression with DNA-derivatized gold nanoparticles, Baptista, {Pedro Miguel Ribeiro Viana}, c}alo Dória Gon{\c, Henriques David, Pereira Eulália, and Franco Ricardo , Journal of Biotechnology, jan, Volume 119, Number 2, p.111–7, (2005) Abstract

Thiol-linked DNA-gold nanoparticles were used in a novel colorimetric method to detect the presence of specific mRNA from a total RNA extract of yeast cells. The method allowed detection of expression of the FSY1 gene that encodes a specific fructose/H+ symporter in Saccharomyces bayanus PYCC 4565. FSY1 is strongly expressed when the yeast is grown in fructose as the sole carbon source, while cells cultivated in glucose as the sole carbon source repress gene expression. The presence of FSY1 mRNA is detected based on color change of a sample containing total RNA extracted from the organism and gold nanoparticles derivatized with a 15-mer of complementary single stranded DNA upon addition of NaCl. If FSY1 mRNA is present, the solution remains pink, changing to blue-purple in the absence of FSY1 mRNA. Direct detection of specific expression was possible from only 0.3 microg of unamplified total RNA without any further enhancement. This novel method is inexpensive, very easy to perform as no amplification or signal enhancement steps are necessary and takes less than 15 min to develop after total RNA extraction. No temperature control is necessary and color change can be easily detected visually.

Using Au-nanoprobes por point-of-need diagnostics of TB., Baptista, Pedro, Veigas {Bruno Miguel Ribeiro}, Portugal Isabel, Couto I., and Viveiros M. , Magazine da Sociedade Portuguesa de Microbiologia, jan, Volume 2012, Number 1, (2012) Abstract

Tuberculosis remains one of the most serious infectious diseases worldwide requiring new tools to circumvent current molecular diagnostics limitations. Nanodiagnostics, i.e. nanotechnology based diagnostics, may do just that by decreasing the time needed for the molecular characterisation of the infecting agent, and allowing for miniaturisation and portability for point-of-need adapted to remote regions without suitable lab equipment.

Cancer nanotechnology - Prospects for cancer diagnostics and therapy, Baptista, {Pedro Viana} , Current Cancer Therapy Reviews, aug, Volume 5, Number 2, p.80–88, (2009) Abstract

Nanotechnology is a multidisciplinary field that brings together diverse fields of research and development such as engineering, biology, physics and chemistry. Formal definitions of nanotechnology refer to man-made devices, components and structures in the 1-100 nm range in at least one dimension. Advances in nanoscience are having a significant impact on many scientific fields, boosting the development of a variety of important technologies. Nanotechnology offers an unprecedented opportunity to interact with cancer cells in real time at the molecular and cellular scale. Because of their small size, nanoscale devices can readily interact with biomolecules on both the surface of cells and inside of cells. The concerted development of nanoscale devices, structures and components have provided essential breakthroughs in monitoring and fighting cancer at the earliest stages of the cancer process. Nanotechnology offers a wealth of tools that may provide researchers with new and innovative ways to diagnose and treat cancer - new imaging agents; systems for real-time assessments of therapeutic and surgical efficacy; multifunctional, targeted devices capable of bypassing biological barriers to deliver multiple therapeutic agents directly to cancer cells and tissues that play a critical role in cancer growth and metastasis; agents that can monitor predictive molecular changes allowing for preventive action against precancerous cells becoming malignant; minimizing costs for multiplex analysis. Nanotechnology, if properly integrated with conventional cancer research, may provide extraordinary prospects towards better diagnosis and effective therapy.

Nanodiagnostics: fast colorimetric method for single nucleotide polymorphism/mutation detection, Baptista, {Pedro Miguel Ribeiro Viana}, and Franco Ricardo , Iet Nanobiotechnology, jan, Volume 1, Number 4, p.53–57, (2007) Abstract

Advances in nanosciences are having a significant impact in many areas of research. The impact of new nanotechnologies has been particularly large in biodiagnostics, where a number of nanoparticle-based assays have been introduced for biomolecules detection. To date, applications of nanoparticles have largely focused on DNA-functionalised gold nanoparticles used as the target-specific probes. These gold nanoparticle-based systems can be used for the detection of specific sequences of DNA (pathogen detection, characterisation of mutation and/or single nucleotide polymorphisms) or RNA (without prior retro-transcription and amplification). Here a rapid and inexpensive nanoparticle-based method for single-base mismatch detection (single nucleotide polymorphism/mutation) in DNA samples is reported. Gold nanoparticles derivatised with thiol modified oligonucleotides complementary to DNA targets - Au-nanoprobes - are used to distinguish fully complementary from mismatched sequences, with a single-base mismatch. The authors have successfully applied this strategy to detect common mutations within the beta-globin gene.

Alloy metal nanoparticles for multicolor cancer diagnostics, Baptista, {Pedro V. }, c}alo Doria Gon{\c, and Conde João , Colloidal Quantum Dots/Nanocrystals for Biomedical Applications VI, (2011) Abstract

Cancer is a multigenic complex disease where multiple gene loci contribute to the phenotype. The ability to simultaneously monitor differential expression originating from each locus results in a more accurate indicator of degree of cancerous activity than either locus alone. Metal nanoparticles have been thoroughly used as labels for in vitro identification and quantification of target sequences. We have synthesized nanoparticles with assorted noble metal compositions in an alloy format and functionalized them with thiol-modified ssDNA (nanoprobes). These nanoprobes were then used for the simultaneous specific identification of several mRNA targets involved in cancer development - one pot multicolor detection of cancer expression. The different metal composition in the alloy yield different {"}colors{"} that can be used as tags for identification of a given target. Following a non-cross-linking hybridization procedure previously developed in our group for gold nanoprobes, these multicolor nanoprobes were used for the molecular recognition of several different targets including differently spliced variants of relevant genes (e.g. gene products involved in chronic myeloid leukemia BCR, ABL, BCR-ABL fusion product). Based on the spectral signature of mixtures, before and after induced aggregation of metal nanoparticles, the correct identification could be made. Further application to differentially quantify expression of each locus in relation to another will be presented. The differences in nanoparticle stability and labeling efficiency for each metal combination composing the colloids, as well as detection capability for each nanoprobe will be discussed. Additional studies will be conducted towards allele specific expression studies.

New insights into the use of magnetic force microscopy to discriminate between magnetic and nonmagnetic nanoparticles, Baptista, {Pedro Miguel Ribeiro Viana} , Nanotechnology, jan, Volume 21, Number 30, p.305706, (2010) Abstract
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Precision nanomedicine in cancer: how far are we from personalization?, Baptista, {Pedro Viana} , Expert Review of Precision Medicine and Drug Development, may, Volume 1, Number 3, p.227–228, (2016) Abstract
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