V. C. Martins

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.

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.

A Portable and Autonomous Magnetic Detection Platform for Biosensing

This paper presents a prototype of a platform for biomolecular recognition detection. The system is based on a magnetoresistive biochip that performs biorecognition assays by detecting magnetically tagged targets. All the electronic circuitry for addressing, driving and reading out signals from spin-valve or magnetic tunnel junctions sensors is implemented using off-the-shelf components. Taking advantage of digital signal processing techniques, the acquired signals are processed in real time and transmitted to a digital analyzer that enables the user to control and follow the experiment through a graphical user interface. The developed platform is portable and capable of operating autonomously for nearly eight hours. Experimental results show that the noise level of the described platform is one order of magnitude lower than the one presented by the previously used measurement set-up. Experimental results also show that this device is able to detect magnetic nanoparticles with a diameter of 250 nm at a concentration of about 40 fM. Finally, the biomolecular recognition detection capabilities of the platform are demonstrated by performing a hybridization assay using complementary and non-complementary probes and a magnetically tagged 20mer single stranded DNA target.

A bacteriophage detection tool for viability assessment of Salmonella cells.

Salmonellosis, one of the most common food and water-borne diseases, has a major global health and economic impact. Salmonella cells present high infection rates, persistence over inauspicious conditions and the potential to preserve virulence in dormant states when cells are viable but non-culturable (VBNC). These facts are challenging for current detection methods. Culture methods lack the capacity to detect VBNC cells, while biomolecular methods (e.g. DNA- or protein-based) hardly distinguish between dead innocuous cells and their viable lethal counterparts. This work presents and validates a novel bacteriophage (phage)-based microbial detection tool to detect and assess Salmonella viability. Salmonella Enteritidis cells in a VBNC physiological state were evaluated by cell culture, flow-cytometry and epifluorescence microscopy, and further assayed with a biosensor platform. Free PVP-SE1 phages in solution showed the ability to recognize VBNC cells, with no lysis induction, in contrast to the minor recognition of heat-killed cells. This ability was confirmed for immobilized phages on gold surfaces, where the phage detection signal follows the same trend of the concentration of viable plus VBNC cells in the sample. The phage probe was then tested in a magnetoresistive biosensor platform allowing the quantitative detection and discrimination of viable and VBNC cells from dead cells, with high sensitivity. Signals arising from 3 to 4 cells per sensor were recorded. In comparison to a polyclonal antibody that does not distinguish viable from dead cells, the phage selectivity in cell recognition minimizes false-negative and false-positive results often associated with most detection methods.

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.

OPTIMIZATION AND INTEGRATION OF MAGNETORESISTIVE SENSORS

This paper addresses challenging issues related to the integration of magnetoresistive (MR) sensors in applications such as magnetic field mapping, magnetic bead detection in microfluidic channels, or biochips. Although sharing the same technological principle for detection (magnetoresistance effect), each application has unique specifications in terms of noise, sensitivity, spatial resolution, electrical robustness or geometric constraints. These differences are of high impact for manufacturing, because some strategies used for sensor optimization compromise the freedom for device architecture.

Implementing a strategy for on-chip detection of cell-free DNA fragments using GMR sensors: A translational application in cancer diagnostics using ALU elements

Cell-free DNA (cfDNA) is foreseen as a promising source for liquid biopsies in cancer diagnostics. Despite its promise, methods available for its evaluation lack in robustness or, in the case of next-generation sequencing (NGS), are extremely sensitive but overly complex for routine operation. In contrast to NGS, integrated lab-on-chip devices offer advantages particularly in terms of automation, cost and speed. These devices, however, have rarely demonstrated the detection of biologically relevant DNA fragments originating from blood. To this end, we present a strategy for the magnetic labeling and detection of cfDNA fragments, using an array of 30 magnetoresistive (MR) sensors integrated in a portable biochip platform. As a proof-of-concept, we selected the fragments ALU115 and ALU247, recently identified as promising cancer targets in cfDNA integrity assessment. This work reveals a rational optimization of the DNA probes design and density at the surface which allowed achieving specific target detection and increased inhibition of unspecific interactions, without the need for blocking agents. The developed strategy is adaptable for the detection of mutations, homologous or truncated sequences such as the case of ALU115 and ALU247, sequences that share great similarity. Upon optimization, the MR sensors detected a concentration of the ALU elements within the picomolar range, showing potential for cfDNA analysis in cancer diagnostics.

Detecting antibody-labeled BCG MNPs using a magnetoresistive biosensor and magnetic labeling technique

Tuberculosis is still a major global health concern, causing the estimated death of 1.5 million people per year and being associated with high morbidity. The development of point-of-care diagnostic tools for tuberculosis is mandatory, especially because the fast and accurate detection of the slow-growing Mycobacterium tuberculosis by the conventional diagnostic tests is difficult.The objective of this work was to develop the first steps to achieve a portable method for the diagnosis of tuberculosis, by a sandwich-immunoassay combined with magnetoresistive biochip technology.With the purpose of conjugating 250 nm streptavidin-coated magnetic nanoparticles with anti- M.tuberculosis biotinylated antibodies, Mycobacteriumbovis Bacillus Calmette-Guérin was used as a surrogate for M. tuberculosis bacteria. After magnetic capture, target bacteria were brought in contact with the surface of the magnetoresistive biochip previously functionalized with a secondary anti-M.tuberculosis antibody. Magnetically labeled cells were detected by an array of spin-valve sensors, which change their electrical resistance in the presence of the fringe field of the magnetic particles. Optimization studies on the efficiency of the magnetic capture and further recognition of the bacteria by the secondary antibody on the biochip surface were conducted. The results on the magnetoresistive biochip showed a clear difference in the signal between specific and control (non-specific) sensors, suggesting the usefulness of this technique as a potential biorecognition tool for the development of a point-of-care diagnostic method for tuberculosis.

Integrated Spintronic Platforms for Biomolecular Recognition Detection

This paper covers recent developments in magnetoresistive based biochip platforms fabricated at INESC‐MN, and their application to the detection and quantification of pathogenic waterborn microorganisms in water samples for human consumption. Such platforms are intended to give response to the increasing concern related to microbial contaminated water sources. The presented results concern the development of biological active DNA chips and protein chips and the demonstration of the detection capability of the present platforms. Two platforms are described, one including spintronic sensors only (spin‐valve based or magnetic tunnel junction based), and the other, a fully scalable platform where each probe site consists of a MTJ in series with a thin film diode (TFD). Two microfluidic systems are described, for cell separation and concentration, and finally, the read out and control integrated electronics are described, allowing the realization of bioassays with a portable point of care unit. The present pla...

Nanotechnology and the Detection of Biomolecular Recognition Using Magnetoresistive Transducers

An integrated electronic biochip platform for the detection of biomolecular recognition is described. It includes the detection module, where labeled target DNA/antibody molecules are magnetically arrayed towards immobilized probes (cDNA/antigen) and where DNA-cDNA hybridization or antibody-antigen interaction is detected. Magnetic nanobeads are used as labels for targets, and magnetic field sensors are used to detect beads presence. The present device holds 256 probe sites in a matrix containing one magnetic tunnel junction and one thin film PIN diode at each node. A microfluidics chamber and a credit card sized electronics board complete the microsystem. Diagnostic applications include the detection of cystic fibrosis relate gene mutations (DNA chip) and the detection of Salmonella and Es-cherichia coli presence in water (immunoassay).