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Sandu, ICA, Schäfer S, Magrini D, Bracci S, Roque ACA.  2012.  Cross-Section and Staining-Based Techniques for Investigating Organic Materials in Painted and Polychrome Works of Art: A Review.. Microscopy and Microanalysis. 18(4):860-875. AbstractWebsite

The article presents a review of the use of cross-section and staining techniques for investigating natural organic materials (mainly proteinaceous and oil-based binders/varnishes) in painted and polychrome artworks, considering the requirements of conservation practice and routine diagnostics. The reviewed literature calls attention to the importance of using cross sections to prepare samples for optical microscopy and to different properties of embedding resins; the most appropriate instrumental conditions for optical microscopy; and the advantages and disadvantages of the most common staining techniques. A few case studies were selected to illustrate the use of autofluorescence (intrinsic fluorescence) and induced fluorescence (using specific staining tests and fluorophore-labeled antibodies) for mapping and identifying organic paint materials in cross sections. New directions of research in cross-section analyses and fluorescence-based techniques for the identification and mapping of artistic materials are presented. The complementary use of different stains on the same cross section, further exploration of intrinsic and induced fluorescence of aged versus fresh materials, and applicability of cross-section observation and staining as complementary methods for assessing the effectiveness of restoration treatments, such as cleaning and consolidation, are discussed in the last section of the article.

Sandu, ICA, Roque ACA, Matteini P, Schäfer S, Agati G, Correia CR, Viana JFFP.  2012.  Fluorescence recognition of proteinaceous binders in works of art by a novel integrated system of investigation. Microscopy Research and Technique. 75(3):316-24. AbstractWebsite

Fluorescence microscopy and microspectrofluorometry are important tools in the characterization and identification of proteins, offering a great range of applications in conservation science. Because of their high selectivity and sensitivity, the combination of these techniques can be exploited for improved recognition and quantification of proteinaceous binders in paintings and polychromed works of art. The present article explores an analytical protocol integrating fluorescence microscopy and fluorometry for both identification and mapping of proteinaceous binders (in particular egg and glues) in paint samples. The study has been carried out on historically accurate reconstructions simulating the structure and composition of tempera and oil paints containing these binders. To assess the spatial distribution of specific proteins within the paint layers, cross-sections from the reconstructions were analyzed by fluorescence imaging after staining with an exogenous fluorophore. Reference fluorescence spectra for each layer were acquired by a multichannel spectral analyzer and compared after Gaussian deconvolution. The results obtained demonstrated the effectiveness of the integrated protocol, highlighting the potential for the use of fluorescent staining coupled with microspectrofluorometry as a routine diagnostic tool in conservation science. The current work creates a set of fully characterized reference samples for further comparison with those from actual works of art.

Santana, SDF, Dhadge VL, Roque ACA.  2012.  Dextran-Coated Magnetic Supports Modified with a Biomimetic Ligand for IgG Purification. ACS Applied Materials and Interfaces. 4(11):5907–5914. AbstractWebsite

extran-coated iron oxide magnetic particles modified with ligand 22/8, a protein A mimetic ligand, were prepared and assessed for IgG purification. Dextran was chosen as the agent to modify the surface of magnetic particles by presenting a negligible level of nonspecific adsorption. For the functionalization of the particles with the affinity ligand toward antibodies, three methods have been explored. The optimum coupling method yielded a theoretical maximum capacity for human IgG calculated as 568 ± 33 mg/g and a binding affinity constant of 7.7 × 104 M–1. Regeneration, recycle and reuse of particles was also highly successful for five cycles with minor loss of capacity. Moreover, this support presented specificity and effectiveness for IgG adsorption and elution at pH 11 directly from crude extracts with a final purity of 95% in the eluted fraction.

Santana, SDF, Pina AS, Roque ACA.  2012.   Immobilization of enterokinase on magnetic supports for the cleavage of fusion proteins. Journal of Biotechnology. 161:378–382. AbstractWebsite

Magnetic nanobiocatalysts for tag cleavage on fusion proteins have been prepared by immobilizing
enterokinase (EK) onto iron oxide magnetic nanoparticles coated with biopolymers. Two different
chemistries have been explored for the covalent coupling of EK, namely carbodiimide (EDC coupling)
and maleimide activation (Sulfo coupling). Upon immobilization, EK initial activity lowered but EDC coupling lead to higher activity retention. Regarding the stability ofthe nanobiocatalysts,thesewere recycled
up to ten times with the greater activity losses observed in the first two cycles. The immobilized EK also
proved to cleave a control fusion protein and to greatly simplify the separation of the enzyme from the
reaction mixture.

Santos, G, Alves C, Pádua AC, Palma S, Gamboa H, Roque ACA.  2019.  An Optimized E-nose for Efficient Volatile Sensing and Discrimination. Proceedings of the 12th International Joint Conference on Biomedical Engineering Systems and Technologies - Volume 1: BIODEVICES. , Prague, Czech Republic AbstractPDF

Electronic noses (E-noses), are usually composed by an array of sensors with different selectivities towards classes of VOCs (Volatile Organic Compounds). These devices have been applied to a variety of fields, including environmental protection, public safety, food and beverage industries, cosmetics, and clinical diagnostics. This work demonstrates that it is possible to classify eleven VOCs from different chemical classes using a single gas sensing biomaterial that changes its optical properties in the presence of VOCs. To accomplish this, an in-house built E-nose, tailor-made for the novel class of gas sensing biomaterials, was improved and combined with powerful machine learning techniques. The device comprises a delivery system, a detection system and a data acquisition and control system. It was designed to be stable, miniaturized and easy-to-handle. The data collected was pre-processed and features and curve fitting parameters were extracted from the original response. A recursive feature selection method was applied to select the best features, and then a Support Vector Machine classifier was implemented to distinguish the eleven distinct VOCs. The results show that the followed methodology allowed the classification of all the VOCs tested with 94.6% (± 0.9%) accuracy.

dos Santos, R, Carvalho AL, Roque ACA.  2017.  Renaissance of protein crystallization and precipitation in biopharmaceuticals purification. Biotechnology Advances. 35:–., Number 1: Elsevier Inc. AbstractWebsite

The current chromatographic approaches used in protein purification are not keeping pace with the increasing biopharmaceutical market demand. With the upstream improvements, the bottleneck shifted towards the downstream process. New approaches rely in Anything But Chromatography methodologies and revisiting former techniques with a bioprocess perspective. Protein crystallization and precipitation methods are already implemented in the downstream process of diverse therapeutic biological macromolecules, overcoming the current chromatographic bottlenecks. Promising work is being developed in order to implement crystallization and precipitation in the purification pipeline of high value therapeutic molecules. This review focuses in the role of these two methodologies in current industrial purification processes, and highlights their potential implementation in the purification pipeline of high value therapeutic molecules, overcoming chromatographic holdups.

dos Santos, R, Iria I, Manuel AM, Leandro AP, Madeira CAC, Gonçalves J, Carvalho AL, Roque ACA.  2020.  Magnetic Precipitation: A New Platform for Protein Purification. Biotechnology Journal. 15(9):2000151.
dos Santos, R, Figueiredo C, Viecinski AC, Pina AS, Barbosa AJM, Roque ACA.  2019.  Designed affinity ligands to capture human serum albumin. Journal of Chromatography A. 1583:88-97. AbstractWebsite

Human serum albumin (HSA) in an important therapeutic agent and disease biomarker, with an increasing market demand. By proteins and drugs that bind to HSA as inspiration, a combinatorial library of 64 triazine-based ligands was rationally designed and screened for HSA binding at physiological conditions. Two triazine-based lead ligands (A3A2 and A6A5), presenting more than 50% HSA bound and high enrichment factors, were selected for further studies. Binding and elution conditions for HSA purification from human plasma were optimized for both ligands. The A6A5 adsorbent yielded a purified HSA sample with 98% purity at 100% recovery yield under mild binding and elution conditions.

Santos, R, Romão MJ, Roque ACA, Carvalho AL.  2021.  Magnetic particles used in a new approach for designed protein crystallization. CrystEngComm. 23(5):1083-1090.
Semeano, ATS, Maffei DF, Palma S, Li RWC, Franco BDGM, Roque ACA, Gruber J.  2018.  Tilapia fish microbial spoilage monitored by a single optical gas sensor. Food Control. 89:72-76. AbstractPDFWebsite

As consumption of fish and fish-based foods increases, non-destructive monitoring of fish freshness also becomes more prominent. Fish products are very perishable and prone to microbiological growth, not always easily detected by organoleptic evaluation. The analysis of the headspace of fish specimens through gas sensing is an interesting approach to monitor fish freshness. Here we report a gas sensing method for monitoring Tilapia fish spoilage based on the application of a single gas sensitive gel material coupled to an optical electronic nose. The optical signals of the sensor and the extent of bacterial growth were followed over time, and results indicated good correlation between the two determinations, which suggests the potential application of this simple and low cost system for Tilapia fish freshness monitoring.