Luís P. Fonseca

Trends in DNA biosensors

Abstract Biosensors have witnessed an escalating interest nowadays, both in the research and commercial fields. Deoxyribonucleic acid (DNA) biosensors (genosensors) have been exploited for their inherent physico-chemical stability and suitability to discriminate different organism strains. The main principle of detection among genosensors relies on specific DNA hybridization, directly on the surface of a physical transducer. This review covers the main DNA immobilization techniques reported so far, new micro- and nanotechnological platforms for biosensing and the transduction mechanisms in genosensors. Clinical applications, in particular, demand large-scale and decentralized DNA testing. New schemes for DNA diagnosis include DNA chips and microfluidics, which couples DNA detection with sample pretreatment under in vivo-like hybridization conditions. Higher sensitivity and specificity may arise from nanoengineered structures, like carbon nanotubes (CNTs) and DNA/protein conjugates. A new platform for universal DNA biosensing is also presented, and its implications for the future of molecular diagnosis are argued.

Horseradish peroxidase: a valuable tool in biotechnology

Peroxidases have conquered a prominent position in biotechnology and associated research areas (enzymology, biochemistry, medicine, genetics, physiology, histo- and cytochemistry). They are one of the most extensively studied groups of enzymes and the literature is rich in research papers dating back from the 19th century. Nevertheless, peroxidases continue to be widely studied, with more than 2000 articles already published in 2002 (according to the Institute for Scientific Information). The importance of peroxidases is emphasised by their wide distribution among living organisms and by their multiple physiological roles. They have been divided into three superfamilies according to their source and mode of action: plant peroxidases, animal peroxidases and catalases. Among all peroxidases, horseradish peroxidase (HRP) has received a special attention and will be the focus of this review. A brief description of the three super-families is included in the first section of this review. In the second section, a comprehensive description of the present state of knowledge of the structure and catalytic action of HRP is presented. The physiological role of peroxidases in higher plants is described in the third section. And finally, the fourth section addresses the applications of peroxidases, especially HRP, in the environmental and health care sectors, and in the pharmaceutical, chemical and biotechnological industries.

The influence of culture conditions on mycelial structure and cellulase production by Trichoderma reesei Rut C-30

The morphology of Trichoderma reesei Rut C-30, during submerged cultivations in shake flask, was examined. The influence of the size inoculum and the composition of the fermentation medium on the morphology and cellulase production was studied. Different inoculum sizes were studied but the significative change in fungus morphology was observed for spores concentration between 10(5) and 10(7) spores/ml (i.e. 10(2) and 10(4) spores/ml in pre-culture medium). In the medium without Tween 80, at low inoculum size, the majority of the pellets were large and well individualized, in contrast, at higher inoculation densities small flocs were obtained, with higher production of soluble protein and higher filter paper activity. It was found that the average pellet size seems to be inversely proportional to the inoculum size. Medium composition, namely Tween 80, also influences the morphology of T. reesei Rut C-30 and enzyme production. The presence of Tween 80 in fermentation medium inhibited the pellet formation of this strain.

Partial purification of penicillin acylase from Escherichia coli in poly(ethylene glycol)-sodium citrate aqueous two-phase systems.

Studies on the partition and purification of penicillin acylase from Escherichia coli osmotic shock extract were performed in poly(ethylene glycol)-sodium citrate systems. Partition coefficient behavior of the enzyme and total protein are similar to those described in other reports, increasing with pH and tie line length and decreasing with PEG molecular weight. However, some selectivity could be attained with PEG 1000 systems and long tie line at pH 6.9. Under these conditions 2.6-fold purification with 83% yield were achieved. Influence of pH on partition shows that is the composition of the system and not the net charge of the enzyme that determines the behaviour in these conditions. Addition of NaCl to PEG 3350 systems significantly increases the partition of the enzyme. Although protein partition also increased, purification conditions were possible with 1.5 M NaCl where 5.7-fold purification and 85% yield was obtained. This was possible due to the higher hydrophobicity of the enzyme compared to that of most contaminants proteins.

Femtomolar limit of detection with a magnetoresistive biochip.

In this paper the biological limit of detection of a spin-valve-based magnetoresistive biochip applied to the detection of 20 mer ssDNA hybridization events is presented. Two reactional variables and their impact on the biomolecular recognition efficiency are discussed. Both the influence of a 250 nm diameter magnetic particle attached to the target molecule during the hybridization event and the effect of a magnetic focusing system in the hybridization of pre-labeled target DNA (assisted hybridization) are addressed. The particles carrying the target molecules are attracted to the probe active sensor sites by applying a 40 mA DC current on U-shaped aluminium current lines. Experiments comparing pre-hybridization versus post-hybridization magnetic labeling and passive versus magnetically assisted hybridization were conducted. The efficiency of a passive hybridization is reduced by about 50% when constrained to the operational conditions (sample volume, reaction time, temperature and magnetic label) of an on-chip real-time hybridization assay. This reduction has shown to be constant and independent from the initial target concentration. Conversely, the presence of the magnetic label improved the limit of detection when a magnetically assisted hybridization was performed. The use of a labeled target focusing system has permitted a gain of three orders of magnitude (from 1 pM down to 1 fM) in the sensitivity of the device, as compared with passive, diffusion-controlled hybridization.

Application of surface response analysis to the optimization of penicillin acylase purification in aqueous two-phase systems

Penicillin acylase purification from an Escherichia coli crude extract using PEG 3350–sodium citrate aqueous two-phase systems (ATPS) was optimized. An experimental design was used to evaluate the influence of PEG, sodium citrate and sodium chloride on the purification parameters. A central composite design was defined centred on the previously found conditions for highest purification from an osmotic shock extract. Mathematical models for the partition coefficient of protein and enzyme, balance of protein and enzyme, yield and purification were calculated and statistically validated. Analysis of the contours of constant response as a function of PEG and sodium citrate concentrations for three different concentrations of NaCl revealed different effects of the three factors on the studied parameters. A maximum purification factor of 6.5 was predicted for PEG 3350, sodium citrate and NaCl concentrations of 15.1, 11.0 and 8.52% respectively. However, under these conditions the predicted yield was 61%. A better compromise between these two parameters can be found by superimposing the contour plots of the purification factor and yield for 10.3% NaCl. A region in the experimental space can be defined where the purification factor is always higher than 5.5 with yields exceeding 80%.

Ultrasonic-assisted enzymatic digestion (USAED) for total elemental determination and elemental speciation: A tutorial

Due to its potential as sample treatment for Analytical Chemistry, the Ultrasonic-Assisted Enzymatic Digestion (USAED) for total elemental determination and elemental speciation is described under the most recent achievements published in literature, focusing on the variables that critically affect the performance of this relatively new sample treatment, such as the type of enzymes or the type of ultrasonic system used for the acceleration of the solid-liquid extraction process. Moreover, analytical chemists are aware of common errors produced in data interpretation concerning USAED. In addition, a guide for the rapid application of this methodology is also provided along with detailed explanations. Finally, future trends regarding USAED are also given and commented.

Variation of penicillin acylase partition coefficient with phase volume ratio in poly(ethylene glycol)-sodium citrate aqueous two-phase systems.

The influence of phase volume ratio on partition and purification of penicillin acylase from Escherichia coli on poly(ethylene glycol)-sodium citrate aqueous two-phase systems was studied. In PEG 1000 systems both partition coefficients of the enzyme and total protein increased with decreasing phase volume ratio. However, in PEG 3350 containing NaCl, penicillin acylase follows a reverse trend, while total protein behaves in the same way. Implications for protein purification designs are discussed.

Biosensors as rapid diagnostic tests for tropical diseases

Effective diagnosis of infectious pathogens is essential for disease identification and subsequent adequate treatment, to prevent drug resistance and to adopt suitable public health interventions for the prevention and control of epidemic outbreaks. Particular situations under which medical diagnostics operate in tropical environments make the use of new easy-to-use diagnostic tools the preferred (or even unique) option. These diagnostic tests and devices, usually based on biosensing methods, are being increasingly exploited as promising alternatives to classical, “heavy” lab instrumentation for clinical diagnosis, allowing simple, inexpensive and point-of-care testing. However, in many developing countries the lack of accessibility and affordability for many commercial diagnostic tests remains a major cause of high disease burden in such regions. We present a comprehensive overview about the problems of conventional medical diagnosis of infectious pathologies in tropical regions, while pointing out new m...Effective diagnosis of infectious pathogens is essential for disease identification and subsequent adequate treatment, to prevent drug resistance and to adopt suitable public health interventions for the prevention and control of epidemic outbreaks. Particular situations under which medical diagnostics operate in tropical environments make the use of new easy-to-use diagnostic tools the preferred (or even unique) option. These diagnostic tests and devices, usually based on biosensing methods, are being increasingly exploited as promising alternatives to classical, “heavy” lab instrumentation for clinical diagnosis, allowing simple, inexpensive and point-of-care testing. However, in many developing countries the lack of accessibility and affordability for many commercial diagnostic tests remains a major cause of high disease burden in such regions. We present a comprehensive overview about the problems of conventional medical diagnosis of infectious pathologies in tropical regions, while pointing out new methods and analytical tools for in-the-field and decentralized diagnosis of current major infectious tropical diseases. The review includes not only biosensor-based rapid diagnostic tests approved by regulatory entities and already commercialized, but also those at the early stages of research.

Magnetoresistive DNA chips

Publisher Summary Magnetoresistance (MR) technology is being successfully applied to biomolecular recognition in different biological contexts. Micron-sized magnetic labels are already successfully used in biomolecular recognition experiments, but smaller magnetic labels that are non-remanent, non-clustering, with low anisotropy and high susceptibility are required. The existing magnetoresistive sensing technology allows the successful detection of single nanometer-sized magnetic labels. However, real biological recognition results with MR biochip prototypes done at INESC and elsewhere are successful only with micron-sized labels (INESC, NRL, U. Bielefeld) and with 250 nm labels (INESC). An important figure of merit when comparing biomolecular recognition detection platforms is the amount of target material that can be detected. The minimum target concentration that can be detected by MR biochip platforms depends intrinsically on label dimension, and the number of target biomolecules attached to the label that can hybridize. MR technology has shown the potential for single molecule process detection, a target not usually within the reach of most of the competing technologies.