Nanoparticle-based biosensors: development and application

Tese dout., Ciências Biotecnológicas, Universidade do Algarve, 2009

Multi-scale lines and edges in V1 and beyond: brightness, object categorization and recognition, and consciousness

In this paper we present an improved model for line and edge detection in cortical area V1. This model is based on responses of simple and complex cells, and it is multi-scale with no free parameters. We illustrate the use of the multi-scale line/edge representation in different processes: visual reconstruction or brightness perception, automatic scale selection and object segregation. A two-level object categorization scenario is tested in which pre-categorization is based on coarse scales only and final categorization on coarse plus fine scales. We also present a multi-scale object and face recognition model. Processing schemes are discussed in the framework of a complete cortical architecture. The fact that brightness perception and object recognition may be based on the same symbolic image representation is an indication that the entire (visual) cortex is involved in consciousness.

Recognition of facial expressions by cortical multi-scale line and edge coding

Empirical studies concerning face recognition suggest that faces may be stored in memory by a few canonical representations. Models of visual perception are based on image representations in cortical area V1 and beyond, which contain many cell layers for feature extraction. Simple, complex and end-stopped cells provide input for line, edge and keypoint detection. Detected events provide a rich, multi-scale object representation, and this representation can be stored in memory in order to identify objects. In this paper, the above context is applied to face recognition. The multi-scale line/edge representation is explored in conjunction with keypoint-based saliency maps for Focus-of-Attention. Recognition rates of up to 96% were achieved by combining frontal and 3/4 views, and recognition was quite robust against partial occlusions.

Application of molecular tools for detection of plant viruses

Grapevine leafroll disease (GLRD) is one of the most important virus diseases of grapevines worldwide, causing major economical impact. The disease has a complex aetiology and currently eleven phloem-limited viruses, termed in general Grapevine leafroll-associated virus (GLRaVs), have been identified. Two of the GLRaVs, GLRaV-1 and GLRaV-3, are included in the European certification scheme of propagation material. However, the flawed notion that GLRaV-3 is more frequent than GLRaV-1 and that all other GLRaVs are possibly not as relevant for GLRD, has until now precluded the development of specific serological and molecular detection assays and limited the scope of molecular characterization of the viruses known to be associated with the disease. Hence, few studies have addressed the phylodynamics of GLRaVs or even characterized the genetic structure of their natural populations. This generalized lack of molecular information, in turn underlie the deficient capacity to detect the viruses. The phylogenetic analyses were conducted on the basis of the heat shock protein 70 homologue (HSP70h) and the coat protein (CP) genes for GLRaV-1 and the HSP70h, the heat shock protein 90 homologue (HSP90h) and the CP genes for GLRaV-5. The data obtained for GLRaV-1 contributed 83 new CP sequences. This information was combined with previous analysis by other authors and used for the production of new polyclonal IgG, capable of detecting CP variants from all the phylogroups observed. Successful testing of this new tool included tissue print immunoblotting (TPIB) and in situ immunoassay (ISIA). The data obtained for GLRaV-5, contributed 61 new CP and 28 new HSP90h gene sequences. Eight phylogenetic groups were identified on the basis of the CP. Characterization of the genetic structure of the isolates revealed a higher diversity than previously reported and allowed the identification of dominant virus variants. For both GLRaV-1 and GLRaV-5, the effect of vegetative propagation on the virus transmission dynamics was addressed.

Modelfest and contrast-interrelation-function data predicted by a retinal model

The automatic implementation of decoders for a visual perception is achieved as follows. The action described by a production rule is realized by means of the decoder in which a pattern of connections coreesponds to that of stimuli. According to "S.Karasawa,(Proc. of CCCT, Vol.5, pp.194-1999, Austin, Texas, August, 2004)", each program mable controllable connection among inputs is realized by a floating gate avalanche injection MOS FET, where inverted signals are used at writing, and the detection of matching between inputs and connections is carried out by using the signal source in which low level signal is provided via comparatively smaller resistance than high level.

Detection of Incipient Thermal Damage Of Carbon Fiber/Epoxy Composites Using Fluorescent Thermal Damage Probes

Thesis (Ph.D.)--University of Washington, 2013

Why is “blindsight” blind? A new perspective on primary visual cortex, recurrent activity and visual awareness

The neuropsychological phenomenon of blindsight has been taken to suggest that the primary visual cortex (V1) plays a unique role in visual awareness, and that extrastriate activation needs to be fed back to V1 in order for the content of that activation to be consciously perceived. The aim of this review is to evaluate this theoretical framework and to revisit its key tenets. Firstly, is blindsight truly a dissociation of awareness and visual detection? Secondly, is there sufficient evidence to rule out the possibility that the loss of awareness resulting from a V1 lesion simply reflects reduced extrastriate responsiveness, rather than a unique role of V1 in conscious experience? Evaluation of these arguments and the empirical evidence leads to the conclusion that the loss of phenomenal awareness in blindsight may not be due to feedback activity in V1 being the hallmark awareness. On the basis of existing literature, an alternative explanation of blindsight is proposed. In this view, visual awareness is a “global” cognitive function as its hallmark is the availability of information to a large number of perceptual and cognitive systems; this requires inter-areal long-range synchronous oscillatory activity. For these oscillations to arise, a specific temporal profile of neuronal activity is required, which is established through recurrent feedback activity involving V1 and the extrastriate cortex. When V1 is lesioned, the loss of recurrent activity prevents inter-areal networks on the basis of oscillatory activity. However, as limited amount of input can reach extrastriate cortex and some extrastriate neuronal selectivity is preserved, computations involving comparison of neural firing rates within a cortical area remain possible. This enables “local” read-out from specific brain regions, allowing for the detection and discrimination of basic visual attributes. Thus blindsight is blind due to lack of “global” long-range synchrony, and it functions via “local” neural readout from extrastriate areas.

Image, video and 3D data registration: medical, satellite and video processing applications with quality metrics

Data registration refers to a series of techniques for matching or bringing similar objects or datasets together into alignment. These techniques enjoy widespread use in a diverse variety of applications, such as video coding, tracking, object and face detection and recognition, surveillance and satellite imaging, medical image analysis and structure from motion. Registration methods are as numerous as their manifold uses, from pixel level and block or feature based methods to Fourier domain methods. This book is focused on providing algorithms and image and video techniques for registration and quality performance metrics. The authors provide various assessment metrics for measuring registration quality alongside analyses of registration techniques, introducing and explaining both familiar and state–of–the–art registration methodologies used in a variety of targeted applications.

Analysis of six fungicides and one acaricide in still and fortified wines using solid-phase microextraction-gas chromatography/tandem mass spectrometry

A multiresidue gas chromatographic method for the determination of six fungicides (captan, chlorthalonil, folpet, iprodione, procymidone and vinclozolin) and one acaricide (dicofol) in still and fortified wines was developed. Solid-phase microextraction (SPME) was chosen for the extraction of the compounds from the studied matrices and tandem mass spectrometry (MS/MS) detection was used. The extraction consists in a solvent free and automated procedure and the detection is highly sensitive and selective. Good linearity was obtained with correlation coefficients of regression (R2) > 0.99 for all the compounds. Satisfactory results of repeatability and intermediate precision were obtained for most of the analytes (RSD < 20%). Recoveries from spiked wine ranged from 80.1% to 112.0%. Limits of quantification (LOQs) were considerably below the proposedmaximumresidue limits (MRLs) for these compounds in grapes and below the suggested limits for wine (MRLs/10), with the exception of captan.

Analysis of polycyclic aromatic hydrocarbons in fish: optimisation and validation of microwave-assisted extraction

An accurate and sensitive method for determination of 18 polycyclic aromatic hydrocarbons (PAHs) (16 PAHs considered by USEPA as priority pollutants, dibenzo[a,l]pyrene and benzo[j]fluoranthene) in fish samples was validated. Analysis was performed by microwave-assisted extraction and liquid chromatography with photodiode array and fluorescence detection. Response surface methodology was used to find the optimal extraction parameters. Validation of the overall methodology was performed by spiking assays at four levels and using SRM 2977. Quantification limits ranging from 0.15–27.16 ng/g wet weight were obtained. The established method was applied in edible tissues of three commonly consumed and commercially valuable fish species (sardine, chub mackerel and horse mackerel) originated from Atlantic Ocean. Variable levels of naphthalene (1.03–2.95 ng/g wet weight), fluorene (0.34–1.09 ng/g wet weight) and phenanthrene (0.34–3.54 ng/g wet weight) were detected in the analysed samples. None of the samples contained detectable amounts of benzo[a]pyrene, the marker used for evaluating the occurrence and carcinogenic effects of PAHs in food.

Assessment of nutritional and metabolic profiles of pea shoots: The new ready-to-eat baby-leaf vegetable

Pea-shoots are a new option as ready-to-eat baby-leaf vegetable. However, data about the nutritional composition and the shelf-life stability of these leaves, especially their phytonutrient composition is scarce. In this work, the macronutrient, micronutrient and phytonutrients profile of minimally processed pea shoots were evaluated at the beginning and at the end of a 10-day storage period. Several physicochemical characteristics (color, pH, total soluble solids, and total titratable acidity) were also monitored. Standard AOAC methods were applied in the nutritional value evaluation, while chromatographic methods with UV–vis and mass detection were used to analyze free forms of vitamins (HPLC-DAD-ESI-MS/MS), carotenoids (HPLC-DAD-APCI-MSn) and flavonoid compounds (HPLC-DAD-ESI-MSn). Atomic absorption spectrometry (HR-CS-AAS) was employed to characterize the mineral content of the leaves. As expected, pea leaves had a high water (91.5%) and low fat (0.3%) and carbohydrate (1.9%) contents, being a good source of dietary fiber (2.1%). Pea shoots showed a high content of vitamins C, E and A, potassium and phosphorous compared to other ready-to-eat green leafy vegetables. The carotenoid profile revealed a high content of β-carotene and lutein, typical from green leafy vegetables. The leaves had a mean flavonoid content of 329 mg/100 g of fresh product, mainly composed by glycosylated quercetin and kaempferol derivatives. Pea shoots kept their fresh appearance during the storage being color maintained throughout the shelf-life. The nutritional composition was in general stable during storage, showing some significant (p < 0.05) variation in certain water-soluble vitamins.

Novel Prostate Specific Antigen plastic antibody designed withcharged binding sites for an improved protein binding and itsapplication in a biosensor of potentiometric transduction

This work shows that the synthesis of protein plastic antibodies tailored with selected charged monomersaround the binding site enhances protein binding. These charged receptor sites are placed over a neutralpolymeric matrix, thus inducing a suitable orientation the protein reception to its site. This is confirmed bypreparing control materials with neutral monomers and also with non-imprinted template. This concepthas been applied here to Prostate Specific Antigen (PSA), the protein of choice for screening prostate can-cer throughout the population, with serum levels >10 ng/mL pointing out a high probability of associatedcancer.Protein Imprinted Materials with charged binding sites (C/PIM) have been produced by surfaceimprinting over graphene layers to which the protein was first covalently attached. Vinylben-zyl(trimethylammonium chloride) and vinyl benzoate were introduced as charged monomers labellingthe binding site and were allowed to self-organize around the protein. The subsequent polymerizationwas made by radical polymerization of vinylbenzene. Neutral PIM (N/PIM) prepared without orientedcharges and non imprinted materials (NIM) obtained without template were used as controls.These materials were used to develop simple and inexpensive potentiometric sensor for PSA. Theywere included as ionophores in plasticized PVC membranes, and tested over electrodes of solid or liq-uid conductive contacts, made of conductive carbon over a syringe or of inner reference solution overmicropipette tips. The electrodes with charged monomers showed a more stable and sensitive response,with an average slope of -44.2 mV/decade and a detection limit of 5.8 × 10−11mol/L (2 ng/mL). The cor-responding non-imprinted sensors showed lower sensitivity, with average slopes of -24.8 mV/decade.The best sensors were successfully applied to the analysis of serum, with recoveries ranging from 96.9to 106.1% and relative errors of 6.8%.

Chitosan/AuNPs Modified Graphene Electrochemical Sensor for Label-Free Human Chorionic Gonadotropin Detection

A new immunosensor is presented for human chorionic gonadotropin (hCG), made by electrodepositing chitosan/gold-nanoparticles over graphene screen-printed electrode (SPE). The antibody was covalently bound to CS via its Fc-terminal. The assembly was controlled by electrochemical Impedance Spectroscopy (EIS) and followed by Fourier Transformed Infrared (FTIR). The hCG-immunosensor displayed linear response against the logarithm-hCG concentration for 0.1–25 ng/mL with limit of detection of 0.016 ng/mL. High selectivity was observed in blank urine and successful detection of hCG was also achieved in spiked samples of real urine from pregnant woman. The immunosensor showed good detection capability, simplicity of fabrication, low-cost, high sensitivity and selectivity.

New molecularly-imprinted polymer for carnitine and its application as ionophore in potentiometric selective membranes

Carnitine (CRT) is a biological metabolite found in urine that contributes in assessingseveral disease conditions, including cancer. Novel quick screening procedures for CRT are therefore fundamental. This work proposes a novel potentiometric device where molecularly imprinted polymers (MIPs) were used as ionophores. The host-tailored sites were imprinted on a polymeric network assembled by radical polymerization of methacrylic acid (MAA) and trimethylpropane trimethacrylate (TRIM). Non-imprinted polymers (NIPs) were produced as control by removing the template from the reaction media. The selective membrane was prepared by dispersing MIP or NIP particles in plasticizer and poly(vinyl chloride), PVC, and casting this mixture over a solid contact support made of graphite. The composition of the selective membrane was investigated with regard to kind/amount of sensory material (MIP or NIP), and the need for a lipophilic additive. Overall, MIP sensors with additive exhibited the best performance, with near-Nernstian response down to ~ 1 × 10− 4 mol L− 1, at pH 5, and a detection limitof ~ 8 × 10− 5 mol L− 1. Suitable selectivity was found for all membranes, assessed by the matched potential method against some of the most common species in urine (urea, sodium, creatinine, sulfate, fructose and hemoglobin). CRT selective membranes including MIP materials were applied successfully to the potentiometric determination of CRT in urine samples.

Improving the early diagnostic of prostate cancer by multiple biomarker detection with new biosensing devices

Prostate cancer (PCa) is the most common form of cancer in men, in Europe (World Health Organization data). The most recent statistics, in Portuguese territory, confirm this scenario, which states that about 50% of Portuguese men may suffer from prostate cancer and 15% of these will die from this condition. Its early detection is therefore fundamental. This is currently being done by Prostate Specific Antigen (PSA) screening in urine but false positive and negative results are quite often obtained and many patients are sent to unnecessary biopsy procedures. This early detection protocol may be improved, by the development of point-of-care cancer detection devices, not only to PSA but also to other biomarkers recently identified. Thus, the present work aims to screen several biomarkers in cultured human prostate cell lines, serum and urine samples, developing low cost sensors based on new synthetic biomaterials. Biomarkers considered in this study are the following: prostate specific antigen (PSA), annexin A3 (ANXA3), microseminoprotein-beta (MSMB) and sarcosine (SAR). The biomarker recognition may occurs by means of molecularly imprinted polymers (MIP), which are a kind of plastic antibodies, and enzymatic approaches. The growth of a rigid polymer, chemically stable, using the biomarker as a template allows the synthesis of the plastic antibody. MIPs show high sensitivity/selectivity and present much longer stability and much lower price than natural antibodies. This nanostructured material was prepared on a carbon solid. The interaction between the biomarker and the sensing-material produces electrical signals generating quantitative or semi-quantitative data. These devices allow inexpensive and portable detection in point-of-care testing.