Single-Particle Plasmon Sensor to Monitor Proteolytic Activity in Real Time.

We have established a label-free plasmonic platform that monitors proteolytic activity in real time. The sensor consists of a random array of gold nanorods that are functionalized with a design peptide that is specifically cleaved by thrombin, resulting in a blueshift of the longitudinal plasmon. By monitoring the plasmon of many individual nanorods, we determined thrombin's proteolytic activity in real time and inferred relevant kinetic parameters. Furthermore, a comparison to a kinetic model revealed that the plasmon shift is dictated by a competition between peptide cleavage and thrombin binding, which have opposing effects on the measured plasmon shift. The dynamic range of the sensor is greater than two orders of magnitude, and it is capable of detecting physiologically relevant levels of active thrombin down to 3 nM in buffered conditions. We expect these plasmon-mediated label-free sensors to open the window to a range of applications stretching from the diagnostic and characterization of bleeding disorders to fundamental proteolytic and pharmacological studies.

Monitoring Proteolytic Activity in Real Time: A New World of Opportunities for Biosensors.

Proteases play a pivotal role in several biological processes, from digestion, cell proliferation, and differentiation to fertility. Deregulation of protease metabolism can result in several pathological conditions (i.e., cancer, neurodegenerative disorders, and others). Therefore, monitoring proteolytic activity in real time could have a fundamental role in the early diagnosis of these diseases. Herein, the main approaches used to develop biosensors for monitoring proteolytic activity are reviewed. A comparison of the advantages and disadvantages of each approach is provided along with a discussion of their importance and promising opportunities for the early diagnosis of severe diseases. This new era of biosensors can be characterized by the ability to control and monitor biological processes, ultimately improving the potential of personalized medicine.

Density Gradient Selection of Colloidal Silver Nanotriangles for Assembling Dye-Particle Plasmophores.

A simple method based on sucrose density gradient centrifugation is proposed here for the fractionation of colloidal silver nanotriangles. This method afforded particle fractions with surface plasmon resonances, spanning from red to infrared spectral ranges that could be used to tune optical properties for plasmonic applications. This feature was exemplified by selecting silver nanotriangle samples with spectral overlap with Atto-655 dye's absorption and emission in order to assemble dye-particle plasmophores. The emission brightness of an individual plasmophore, as characterized by fluorescence correlation spectroscopy, is at least 1000-fold more intense than that of a single Atto-655 dye label, which renders them as promising platforms for the development of fluorescence-based nanosensors.

Integrated Optical Mach-Zehnder Interferometer Based on Organic-Inorganic Hybrids for Photonics-on-a-Chip Biosensing Applications.

The development of portable low-cost integrated optics-based biosensors for photonics-on-a-chip devices for real-time diagnosis are of great interest, offering significant advantages over current analytical methods. We report the fabrication and characterization of an optical sensor based on a Mach-Zehnder interferometer to monitor the growing concentration of bacteria in a liquid medium. The device pattern was imprinted on transparent self-patternable organic-inorganic di-ureasil hybrid films by direct UV-laser, reducing the complexity and cost production compared with lithographic techniques or three-dimensional (3D) patterning using femtosecond lasers. The sensor performance was evaluated using, as an illustrative example, E. coli cell growth in an aqueous medium. The measured sensitivity (2 × 10-4 RIU) and limit of detection (LOD = 2 × 10-4) are among the best values known for low-refractive index contrast sensors. Furthermore, the di-ureasil hybrid used to produce this biosensor has additional advantages, such as mechanical flexibility, thermal stability, and low insertion losses due to fiber-device refractive index mismatch (~1.49). Therefore, the proposed sensor constitutes a direct, compact, fast, and cost-effective solution for monitoring the concentration of lived-cells.

Metabolomics of silver nanoparticles toxicity in HaCaT cells: structure-activity relationships and role of ionic silver and oxidative stress.

The widespread use of silver nanoparticles (AgNPs) is accompanied by a growing concern regarding their potential risks to human health, thus calling for an increased understanding of their biological effects. The aim of this work was to systematically study the extent to which changes in cellular metabolism were dependent on the properties of AgNPs, using NMR metabolomics. Human skin keratinocytes (HaCaT cells) were exposed to citrate-coated AgNPs of 10, 30 or 60 nm diameter and to 30 nm AgNPs coated either with citrate (CIT), polyethylene glycol (PEG) or bovine serum albumin (BSA), to assess the influence of NP size and surface chemistry. Overall, CIT-coated 60 nm and PEG-coated 30 nm AgNPs had the least impact on cell viability and metabolism. The role of ionic silver and reactive oxygen species (ROS)-mediated effects was also studied, in comparison to CIT-coated 30 nm particles. At concentrations causing an equivalent decrease in cell viability, Ag(+ )ions produced a change in the metabolic profile that was remarkably similar to that seen for AgNPs, the main difference being the lesser impact on the Krebs cycle and energy metabolism. Finally, this study newly reported that while down-regulated glycolysis and disruption of energy production were common to AgNPs and H2O2, the impact on some metabolic pathways (GSH synthesis, glutaminolysis and the Krebs cycle) was independent of ROS-mediated mechanisms. In conclusion, this study shows the ability of NMR metabolomics to define subtle biochemical changes induced by AgNPs and demonstrates the potential of this approach for rapid, untargeted screening of pre-clinical toxicity of nanomaterials in general.

Toward the definition of a peptidome signature and protease profile in chronic periodontitis.

PURPOSE: Chronic periodontitis (CP) is a complex immuno-inflammatory disease that results from preestablished gingivitis. We investigated potential differences in salivary peptidome in health and CP. EXPERIMENTAL DESIGN: Saliva was collected from nine CP patients and ten healthy subjects, from which five CP and five healthy were enriched following endoProteoFASP approach, separated and identified by nanoHPLC-MALDI-TOF/TOF. Protease prediction was carried out in silico with Proteasix. Parallel gelatin and collagen (I) zymographies were performed to study proteolytic activity in CP. RESULTS: An association of CP with increased gelatinolytic and collagenolytic activity was observed, which is mainly attributed to metalloproteases, remarkably MMP9. Protease prediction revealed distinct protease profiles in CP and in health. Peptidomic data corroborated the inflammatory status, and demonstrated that intact histatin 1 may play an important role in the defense response against oral pathogens. The application of the endoProteoFASP approach to study the salivary peptidome of CP subjects resulted in the identification of eight surrogate peptide markers, which may be used in multiplex to identify CP. These peptides belong to acidic PRP and to P-B peptide. Particularly, P-B peptide fragments exhibited domains with potential predicted antimicrobial activity, corroborating an antimicrobial function. CONCLUSIONS AND CLINICAL RELEVANCE: The comparison between the salivary peptidome obtained by control and CP samples showed a specific association of eight peptides to CP, with remarkable predicted antimicrobial activity, which should be further validated in studies with large number of subjects.

Magnetic chelating nanoprobes for enrichment and selective recovery of metalloproteases from human saliva.

New magnetic nanoprobes based on surface-functionalized magnetite particles were synthesized and used to selectively capture metalloproteases from human saliva samples. In addition to their high specific surface area, these nanoparticles have metal ion chelating moieties grafted on their surface by the reaction with the organosilane EDTA-TMS ((N-(trimethoxysilylpropyl)-ethylenediaminetriacetate trisodium salt). The most distinct feature of these particles is their capability to selectively recover metalloproteases even in highly diluted saliva samples. The high affinity of these materials for metalloproteases was attributed to the formation of chelates between the chelating moieties and the metal ions of metalloproteases. These magnetic particles exhibited high colloidal stability in the biological standard buffers MES, HEPES, TEAB and Tris-HCl, within a significant pH range (pH 5-9) and, due to their magnetic features, enabled a fast separation of the metalloproteases from the medium by simply applying an external magnetic field. Thus, these materials were proven to be valuable probes for the selective enrichment and rapid recovery of metalloproteases from human saliva, particularly when dealing with trace amounts of material.

Glycoprotein enrichment method using a selective magnetic nano-probe platform (MNP) functionalized with lectins.

Protein post-translational modifications (PTMs) have increasingly become a research field of incredible importance to fully understand the regulation of biological processes in health and disease. Among PTMs, glycosylation is one of the most studied for which contributed the development and improvement of enrichment techniques. Nowadays, glycoprotein enrichment methods are based on lectin affinity, covalent interactions, and hydrophilic interaction liquid chromatography (HILIC). Nonetheless, the nanotechnology era has fetched new methods to enrich glycoproteins from complex samples as human biological fluids. For instance, magnetic nanoparticles (MNPs) are being used as an interesting enrichment approach allowing a better characterization of glycoproteins and glycopeptides.In this chapter, we describe an enrichment method based on MNPs functionalized with lectins (Concavalin A, wheat germ agglutinin, and Maackia amurensis lectin) to enrich specific sets of glycoproteins from biological fluids. Moreover, it is proposed a bioinformatic strategy to deal with data retrieved from mass spectrometry analysis of enriched samples aiming the identification of relevant biological processes modulated by a given stimuli and, ultimately, of new biomarkers for disease screening/management.

Bionanoconjugation for proteomics applications - An overview.

Formed as an interdisciplinary domain on the basis of Human Genome Project, Proteomics aims at the large-scale study of proteins. The enthusiasm that resulted from obtaining the complete human genetic information has, however, been chastened by the realization that this information contributes little to the comprehension and knowledge of the expressed proteins. In the wake of this realization, the Human Proteome Project (HUPO) was founded, which is a global, collaborative initiative, aiming at the complete characterization of the proteins of all protein-coding genes. Nonetheless, the rapid detection of these molecules in complex biological samples under conditions considered to be of clinical relevance is extremely difficult, requiring the development of very sensitive, robust, reproducible and high throughput platforms. Nanoproteomics has emerged as a feasible, promising option, offering short assay times, low sample consumption, ultralow detection and high throughput capacity. Additionally, the successful synthesis of biomolecules and nanoparticle hybrids yields systems which often exhibit new or improved features. Herein, we overview the recent advances in bioconjugation at the nanolevel and, specifically, their application in Proteomics, discussing not only the merits and prospects of Proteomics, but also present day limitations.