BioCode gold-nanobeacon for the detection of fusion transcripts causing chronic myeloid leukemia

This work was supported by Fundacao para a Ciencia e Tecnologia, Ministry of Science and Education (FCT/MEC) [PTDC/QUI-QUI/112597/2009; PTDC/BBB-NAN/1812/2012; CIGMH (PEST-OE/SAU/UI0009/2011); SFRH/BD/87836/2012 to M.C. and SFRH/BPD/88322/2012 to L.G.]; and by Unidade de Ciencias Biomoleculares Aplicadas-UCIBIO [(UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728)].BACKGROUND: Gold-nanobeacons (Au-nanobeacons) have proven to be versatile systems for molecular diagnostics and therapeutic actuators. Here, we present the development and characterization of two gold nanobeacons combined with Förster resonance energy transfer (FRET) based spectral codification for dual mode sequence discrimination. This is the combination of two powerful technologies onto a single nanosystem. RESULTS: We proved this concept to detect the most common fusion sequences associated with the development of chronic myeloid leukemia, e13a2 and e14a2. The detection is based on spectral shift of the donor signal to the acceptor, which allows for corroboration of the hybridization event. The Au-nanobeacon acts as scaffold for detection of the target in a homogenous format whose output capability (i.e. additional layer of information) is potentiated via the spectral codification strategy. CONCLUSIONS: The spectral coded Au-nanobeacons permit the detection of each of the pathogenic fusion sequences, with high specificity towards partial complementary sequences. The proposed BioCode Au-nanobeacon concept provides for a nanoplatform for molecular recognition suitable for cancer diagnostics.publishersversionpublishe

On the CFD Analysis of a Stratified Taylor-Couette System Dedicated to the Fabrication of Nanosensors

Since the pioneering work of Taylor, the analysis of flow regimes of incompressible, viscous fluids contained in circular Couette systems with independently rotating cylinders have charmed many researchers. The characteristics of such kind of flows have been considered for some industrial applications. Recently, Taylor-Couette flows found an innovative application in the production of optical fiber nanotips, to be used in molecular biology and medical diagnostic fields. Starting from the activity of Barucci et al., the present work concerns the numerical analysis of a Taylor-Couette system composed by two coaxial counter-rotating cylinders with low aspect ratio and radius ratio, filled with three stratified fluids. An accurate analysis of the flow regimes is performed, considering both the variation of inner and outer rotational speed and the reduction of fiber radius due to etching process. The large variety of individuated flow configurations provides useful information about the possible use of the Taylor-Couette system in a wide range of engineering applications. For the present case, the final objective is to provide accurate information to manufacturers of fiber nanotips about the expected flow regimes, thus helping them in the setup of the control process that will be used to generate high-quality products

Sensores de fibra ótica como tecnologia não-invasiva para avaliação da pressão arterial central

Doutoramento em Engenharia FísicaCom o presente trabalho pretendeu-se explorar soluções de fibra ótica na aquisição da onda de pulso na artéria carótida, para análise da sua morfologia e cálculo da pressão arterial central. Foram desenvolvidos três sistemas, dois baseados em redes de Bragg, gravadas em fibra de sílica, e outro em modulação de intensidade, usando fibra ótica de plástico. O primeiro sensor foi desenvolvido com o objetivo de testar a exequibilidade da utilização de fibra ótica nesta aplicação. Após resultados promissores da sua caracterização e testes em sujeitos, o desenvolvimento dos dois sensores consequentes teve por objetivo o aumento da sensibilidade e facilidade de utilização das sondas, pela melhoria da sua forma, portabilidade e autonomia. A solução baseada em intensidade mostrou-se ainda como um sistema que coligava desempenho a baixo custo, tendo por isso sido submetida a um estudo pré-clínico, comparando o seu desempenho ao de um dispositivo comercial, de natureza eletromecânica, numa pequena coorte de indivíduos saudáveis. Este estudo teve como objetivo investigar a correlação dos resultados obtidos com a sonda de fibra ótica e o dispositivo comercial. Tendo-se obtido uma correlação muito forte entre as duas técnicas, o dispositivo foi proposto para avaliação clínica. O desempenho da sonda foi assim comparado a um dispositivo comercial, numa coorte de indivíduos hipertensos. Foram também levados a cabo testes invasivos, usando como referência ondas de pressão obtidas no lúmen da artéria aorta em contexto de cateterismo cardíaco. Em ambos os estudos clínicos foram obtidos coeficientes de correlação muito fortes e diferenças de pressão média na gama obtida para dispositivos comerciais. Conclui-se assim que o dispositivo baseado em modulação de intensidade surge como uma promissora alternativa de baixo custo aos dispositivos eletromecânicos de avaliação de pressão arterial central disponíveis no mercado.The present study aimed to explore fibre optic solutions in the acquisition of the pulse wave in the carotid artery, to its morphology analysis and central arterial pressure calculation. Three systems were developed, two based on Bragg gratings, engraved in silica fibre, and another on intensity modulation, using plastic optical fibre. The first sensor was developed in order to test the feasibility of the fibre optics use in this application. After promising results in the characterization and small tests in subjects, the development of the consequent two sensors had as main goals increasing the probes sensitivity and user-friendliness, by improving its shape, portability and autonomy. The intensity-based solution proved to be the system that best combined performance to low cost, and thus was subjected to a pre-clinical study, comparing its performance to a commercial device in a small cohort of healthy individuals. This study aimed to investigate the correlation between the results obtained with the fibre optic probe and the commercial device. Having been obtained a very strong correlation between the two techniques, the device was proposed for clinical evaluation. The probe’s performance was therefore compared to a non-invasive commercial device, in a cohort of hypertensive individuals. Invasive testing was also performed, using as reference pressure waves obtained in the lumen of the aortic artery in cardiac catheterization context. In both trials very strong correlation coefficients were obtained, as well as medium pressure differences in the range verified for commercial devices. It is concluded that the device based on intensity modulation arises as promising low cost alternative to central arterial pressure assessment electromechanical devices available in the market

CNT MEMBRANE PLATFORMS FOR TRANSDERMAL DRUG DELIVERY AND APTAMER MODULATED TRANSPORT

CNT membrane platforms are biomimetic polymeric membranes imbedded with carbon nanotubes which show fast fluid flow, electric conductivity, and the ability to be grafted with chemistry. A novel micro-dialysis probe nicotine concentration sampling technique was proposed and proved in vitro, which could greatly improve the efficiency and accuracy of future animal transdermal studies. To enhance the scope of transdermal drug delivery which was limited to passive diffusion of small, potent lipophilic drugs, a wire mesh lateral electroporation design was also proposed which could periodically disrupt the skin barrier and enhance drug flux. It was shown that AMP binding aptamer at the tip of carbon nanotubes may act as gatekeepers and regulate ionic transport through CNT membrane. Multiple cycle gating of ionic transport upon AMP binding/unbinding which changes the aptamer conformation was displayed. This CNT membrane-aptamer system closely mimics how protein ion channels modulate ion flow by responding to stimuli, which may have significant impact on active membrane transport. Finally an enhanced electroosmosis concept by “ratchet” functionalization at both ends of carbon nanotubes in was discussed. Direct observation of water transport by electroosmosis was made possible through enhanced flow in vertically aligned high flux CNT membranes

Optical Fiber Nanotips Coated with Molecular Beacons for DNA Detection

Optical fiber sensors, thanks to their compactness, fast response and real-time measurements, have a large impact in the fields of life science research, drug discovery and medical diagnostics. In recent years, advances in nanotechnology have resulted in the development of nanotools, capable of entering the single cell, resulting in new nanobiosensors useful for the detection of biomolecules inside living cells. In this paper, we provide an application of a nanotip coupled with molecular beacons (MBs) for the detection of DNA. The MBs were characterized by hybridization studies with a complementary target to prove their functionality both free in solution and immobilized onto a solid support. The solid support chosen as substrate for the immobilization of the MBs was a 30 nm tapered tip of an optical fiber, fabricated by chemical etching. With this set-up promising results were obtained and a limit of detection (LOD) of 0.57 nM was reached, opening up the possibility of using the proposed nanotip to detect mRNAs inside the cytoplasm of living cells