Publications in the Year: 2014

Journal Article

Carlos, FF, Flores O, Doria G, Baptista PV.  2014.  Characterization of genomic SNP via colorimetric detection using a single gold nanoprobe. Analytical Biochemistry. 465:1-5. AbstractWebsite

Identification of specific nucleic acid sequences mediated by gold nanoparticles derivatized thiol-modified oligonucleotides (Au-nanoprobes) has been proven to be a useful tool in molecular diagnostics. Here, we demonstrate that, on optimization, detection may be simplified via the use of a single Au-nanoprobe to detect a single nucleotide polymorphism (SNP) in homo- or heterozygote condition. We validated this non-cross-linking approach through the analysis of 20 clinical samples using a single specific Au-nanoprobe for an SNP in the FTO (fat mass and obesity-associated) gene against direct DNA sequencing. Sensitivity, specificity, and limit of detection (LOD) were determined, and statistical differences were calculated by one-way analysis of variance (ANOVA) and a post hoc Tukey's test to ascertain whether there were any differences between Au-nanoprobe genotyped groups. For the first time, we show that the use of a single Au-nanoprobe can detect SNP for each genetic status (wild type, heterozygous, or mutant) with high degrees of sensitivity (87.50%) and specificity (91.67%).

Luis, DV, Silva J, Tomaz AI, de Almeida RFM, Larguinho M, Baptista PV, Martins LMDRS, Silva TFS, Borralho PM, Rodrigues CMP, Rodrigues AS, Pombeiro AJL, Fernandes AR.  2014.  Insights into the mechanisms underlying the antiproliferative potential of a Co(II) coordination compound bearing 1,10-phenanthroline-5,6-dione: DNA and protein interaction studies. Journal of Biological Inorganic Chemistry. 19:787-803., Number 6 Abstract

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Restani, RB, Conde J, Baptista PV, Cidade MT, Bragança AM, Morgado J, Correia IJ, Aguiar-Ricardo A, Bonifacio VDB.  2014.  Polyurea dendrimer for efficient cytosolic siRNA delivery. RSC ADVANCES. 4(97):54872. AbstractWebsite

The design of small interfering RNA (siRNA) delivery materials showing efficacy in vivo is at the forefront of nanotherapeutics research. Polyurea (PURE-type) dendrimers are ‘smart’ biocompatible 3D polymers that unveil a dynamic and elegant back-folding mechanism involving hydrogen bonding between primary amines at the surface and tertiary amines and ureas at the core. Similarly, to a biological proton pump, they are able to automatically and reversibly transform their conformation in response to pH stimulus. Here, we show that PURE-G4 is a useful gene silencing platform showing no cellular toxicity. As a proof of concept we investigated the PURE-G4-siRNA dendriplex, which was shown to be an attractive platform with high transfection efficacy. The simplicity associated with the complexation of siRNA with polyurea dendrimers makes them a powerful tool for efficient cytosolic siRNA delivery.

Veigas, B, Fernandes AR, Baptista PV.  2014.  AuNPs for identification of molecular signatures of resistance. Frontiers in Microbiology. 5 Abstract

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Cabral, RM, Baptista PV.  2014.  Anti-cancer precision theranostics: a focus on multifunctional gold nanoparticles. Expert Review of Molecular Diagnostics. 14:1041-1052., Number 8 Abstract

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Baptista, PV.  2014.  Nanodiagnostics: leaving the research lab to enter the clinics? Diagnosis. 1(4):305–309. AbstractWebsite

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?

Larguinho, M, Correia D, Diniz MS, Baptista PV.  2014.  Evidence of one-way flow bioaccumulation of gold nanoparticles across two trophic levels. Journal of Nanoparticle Research. 16, Number 8 Abstract

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Baptista, PV.  2014.  Gold nanobeacons: A potential nanotheranostics platform. Nanomedicine. 9(15):2247-50.Website
Veigas, B, Branquinho R, Pinto JV, Wojcik PJ, Martins R, Fortunato E, Baptista PV.  2014.  Ion sensing (EIS) real-time quantitative monitorization of isothermal DNA amplification. Biosens Bioelectron. 52:50-5. AbstractWebsite

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Bernacka-Wojcik, I, Ribeiro S, Wojcik PJ, Alves PU, Busani T, Fortunato E, Baptista PV, Covas JA, Aguas H, Hilliou L, Martins R.  2014.  Experimental optimization of a passive planar rhombic micromixer with obstacles for effective mixing in a short channel length. Rsc Advances. 4:56013-56025., Number 99 Abstract

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Martins, P, Rosa D, Fernandes AR, Baptista PV.  2014.  Nanoparticle Drug Delivery Systems: Recent Patents and Applications in Nanomedicine. Recent Patents on Nanomedicine. 3(2):105-118. AbstractWebsite

Traditional methods of drug delivery present several drawbacks, mainly due to off-target effects that may originate severe side and toxic effect to healthy tissues. Parallel to the development of novel more effective drugs, particular effort has been dedicated to develop and optimize drug delivery vehicles capable of specifically targeting the required tissue/organ and to deliver the cargo only where and when it is needed. New drug delivery systems based on nanoscale devices showing new and improved pharmacokinetic and pharmacodynamics properties like enhanced bioavailability, high drug loading or systemic stability have surged in the past decade as promising solutions to the required therapeutic efficacy. Amongst these nanoscale vectors, nanoparticles for drug delivery, such as polymeric, lipidbased, ceramic or metallic nanoparticles, have been at the forefront of pharmaceutical development. The interest in nanomedicine for treatment and diagnosis is clearly reflected on the increasing number of publications and issued patents every year. Here, we provide a broad overview of novel nanoparticle based drug delivery systems, ranging from polymeric systems to metal nanoparticles, while simultaneously listing the most relevant related patents.

Larguinho, M, Cordeiro A, Diniz MS, Costa PM, Baptista PV.  2014.  Metabolic and histopathological alterations in the marine bivalve Mytilus galloprovincialis induced by chronic exposure to acrylamide. Environmental Research. 135:55-62. Abstract

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Bernacka-Wojcik, I, Ribeiro S, Wojcik PJ, Alves PU, Busani T, Fortunato E, Baptista PV, Covas JA, Águas H, Hilliou L, Martins R.  2014.   Experimental optimization of a passive planar rhombic micromixer with obstacles for effective mixing in a short channel length. RSC ADVANCES. 4(99) AbstractWebsite

This paper presents the performance of a passive planar rhombic micromixer with diamond-shaped obstacles and a rectangular contraction between the rhombi. The device was experimentally optimized using water for high mixing efficiency and a low pressure drop over a wide range of Reynolds numbers (Re = 0.1–117.6) by varying geometrical parameters such as the number of rhombi, the distance between obstacles and the contraction width. Due to the large amount of data generated, statistical methods were used to facilitate and improve the results of the analysis. The results revealed a rank of factors influencing mixing efficiency: Reynolds number > number of rhombi > contraction width > inter-obstacles distance. The pressure drop measured after three rhombi depends mainly on Re and inter-obstacle distance. The resulting optimum geometry for the low Re regime has a contraction width of 101 μm and inter-obstacles distance of 93 μm, while for the high Re regime a contraction width of 400 μm and inter-obstacle distance of 121 μm are more appropriate. These mixers enabled 80% mixing efficiency creating a pressure drop of 6.0 Pa at Re = 0.1 and 5.1 × 104 Pa at Re = 117.6, with a mixer length of 2.5 mm. To the authors' knowledge, the developed mixer is one of the shortest planar passive micromixers reported to date.

Roma-Rodrigues, C, Fernandes AR, Baptista PV.  2014.  Exosome in Tumour Microenvironment: Overview of the Crosstalk between Normal and Cancer Cells. Biomed Research International. Abstract

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Baptista, PV.  2014.  3h Gold nanobeacons: a potential nanotheranostics platform. Nanomedicine. 9:2247-2250., Number 15 Abstract

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Conde, J, Larguinho M, Cordeiro A, Raposo LR, Costa PM, Santos S, Diniz M, Fernandes AR, Baptista PV.  2014.  Gold-Nanobeacons for gene therapy: evaluation of genotoxicity, cell toxicity and proteome profiling analysis. Nanotoxicology. 8(5):521-532. AbstractWebsite

Antisense therapy is a powerful tool for post-transcriptional gene silencing suitable for down-regulating target genes associated to disease. Gold nanoparticles have been described as effective intracellular delivery vehicles for antisense oligonucleotides providing increased protection against nucleases and targeting capability via simple surface modification. We constructed an antisense gold-nanobeacon consisting of a stem-looped oligonucleotide double-labelled with 3′-Cy3 and 5′-Thiol-C6 and tested for the effective blocking of gene expression in colorectal cancer cells. Due to the beacon conformation, gene silencing was directly detected as fluorescence increases with hybridisation to target, which can be used to assess the level of silencing. Moreover, this system was extensively evaluated for the genotoxic, cytotoxic and proteomic effects of gold-nanobeacon exposure to cancer cells. The exposure was evaluated by two-dimensional protein electrophoresis followed by mass spectrometry to perform a proteomic profile and 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, glutathione-S-transferase assay, micronucleus test and comet assay to assess the genotoxicity. This integrated toxicology evaluation showed that the proposed nanotheranostics strategy does not exhibit significant toxicity, which is extremely relevant when translating into in vivo systems.

Costa, MN, Veigas B, Jacob JM, Santos DS, Gomes J, Baptista PV, Martins R, Inacio J, Fortunato E.  2014.  A low cost, safe, disposable, rapid and self-sustainable paper-based platform for diagnostic testing: lab-on-paper. Nanotechnology. 25, Number 9 Abstract

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Costa, M, Veigas B, Jacob J, Santos D, Gomes J, Baptista PV, Martins R, Inácio J, Fortunato E.  2014.  Low cost, safe, disposable, rapid and self-sustainable paper-based platform for diagnostic testing - Lab-on-Paper. NANOTECHNOLOGY. 9(25):094006. AbstractWebsite

There is a strong interest in the use of biopolymers in the electronic and biomedical industries, mainly towards low-cost applications. The possibility of developing entirely new kinds of products based on cellulose is of current interest, in order to enhance and to add new functionalities to conventional paper-based products. We present our results towards the development of paper-based microfluidics for molecular diagnostic testing. Paper properties were evaluated and compared to nitrocellulose, the most commonly used material in lateral flow and other rapid tests. Focusing on the use of paper as a substrate for microfluidic applications, through an eco-friendly wax-printing technology, we present three main and distinct colorimetric approaches: (i) enzymatic reactions (glucose detection); (ii) immunoassays (antibodies anti-Leishmania detection); (iii) nucleic acid sequence identification (Mycobacterium tuberculosis complex detection). Colorimetric glucose quantification was achieved through enzymatic reactions performed within specific zones of the paper-based device. The colouration achieved increased with growing glucose concentration and was highly homogeneous, covering all the surface of the paper reaction zones in a 3D sensor format. These devices showed a major advantage when compared to the 2D lateral flow glucose sensors, where some carryover of the coloured products usually occurs. The detection of anti-Leishmania antibodies in canine sera was conceptually achieved using a paper-based 96-well enzyme-linked immunosorbent assay format. However, optimization is still needed for this test, regarding the efficiency of the immobilization of antigens on the cellulose fibres. The detection of Mycobacterium tuberculosis nucleic acids integrated with a non-cross-linking gold nanoprobe detection scheme was also achieved in a wax-printed 384-well paper-based microplate, by the hybridization with a species-specific probe. The obtained results with the above-mentioned proof-of-concept sensors are thus promising towards the future development of simple and cost-effective paper-based diagnostic devices.

Reimão-Pinto, MM, Cordeiro A, Almeida C, Pinheiro AV, Moro A, Lima JC, Baptista PV.  2014.  Dual-color control of nucleotide polymerization sensed by a fluorescence actuator. Photochem Photobiol Sci. 13:751-6., Number 5 AbstractWebsite

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Baptista, PV.  2014.  Nanodiagnostics: leaving the research lab to enter the clinics? Diagnosis (Berl). 1:305-309., Number 4 AbstractWebsite

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Larguinho, M, Costa PM, Sousa G, Costa MH, Diniz MS, Baptista PV.  2014.  Histopathological findings on Carassius auratus hepatopancreas upon exposure to acrylamide: correlation with genotoxicity and metabolic alterations.. Journal of Applied Toxicology . 34(12)
Conde, J, Bao C, Cui D, Baptista PV, Tian F.  2014.  Antibody-drug gold nanoantennas with Raman spectroscopic fingerprints for in vivo tumour theranostics. Journal of Controlled Release. 183:87-93. Abstract

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Pedrosa, P, Veigas B, Machado D, Couto I, Viveiros M, Baptista PV.  2014.  Gold nanoprobes for multi loci assessment of multi-drug resistant tuberculosis. Tuberculosis. 94(3):332-337.: Elsevier AbstractWebsite

Tuberculosis, still one of the leading human infectious diseases, reported 8.7 million new cases in 2011 alone. Also, the increasing rate of multidrug-resistant tuberculosis (MDRTB) and its treatment difficulties pose a serious public health threat especially in developing countries. Resistance to isoniazid and rifampicin, first line antibiotics, is commonly associated with point mutations in katG, inhA and rpoB genes of Mycobacterium tuberculosis complex (MTBC). Therefore, the development of cheap, fast and simple molecular methods to assess susceptibility profiles would have a huge impact in the capacity of early diagnosis and treatment of MDRTB.

Gold nanoparticles functionalized with thiol-modified oligonucleotides (Au-nanoprobes) have shown the potential to provide a rapid and sensitive detection method for MTBC and single base mutations associated with antibiotic resistance, namely the characterization of the three most relevant codons in rpoB gene associated to rifampicin resistance. Here we extend the Au-nanoprobe approach towards discriminating specific mutations within inhA and rpoB genes in PCR amplified DNA from isolates. Using a multiplex PCR reaction for these two genes, it is possible to assess both loci in parallel, and extend the potential of the Au-nanoprobe method to MDRTB molecular characterization with special application in the most frequent Portuguese genotypes.

Quaresma, P, Osorio I, Doria G, Carvalho PA, Pereira A, Langer J, Araujo JP, Pastoriza-Santos I, Liz-Marzan LM, Franco R, Baptista PV, Pereira E.  2014.  Star-shaped magnetite@gold nanoparticles for protein magnetic separation and SERS detection. Rsc Advances. 4:3659-3667., Number 8 AbstractWebsite

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Bao, C, Conde J, Polo E, del Pino P, Moros M, Baptista PV, Grazu V, Cui D, de la Fuente JM.  2014.  A promising road with challenges: where are gold nanoparticles in translational research? Nanomedicine. 9:2353-2370., Number 15 Abstract

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