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Palma, S, Traguedo AP, Porteira AR, Frias MJ, Gamboa H, Roque ACA.  2018.  Machine learning for the meta-analyses of microbial pathogens’ volatile signatures. Scientific Reports. 8:3360. Abstractdataset and scripts PDFWebsite

Non-invasive and fast diagnostic tools based on volatolomics hold great promise in the control of infectious diseases. However, the tools to identify microbial volatile organic compounds (VOCs) discriminating between human pathogens are still missing. Artificial intelligence is increasingly recognised as an essential tool in health sciences. Machine learning algorithms based in support vector machines and features selection tools were here applied to find sets of microbial VOCs with pathogen-discrimination power. Studies reporting VOCs emitted by human microbial pathogens published between 1977 and 2016 were used as source data. A set of 18 VOCs is sufficient to predict the identity of 11 microbial pathogens with high accuracy (77%), and precision (62–100%). There is one set of VOCs associated with each of the 11 pathogens which can predict the presence of that pathogen in a sample with high accuracy and precision (86–90%). The implemented pathogen classification methodology supports future database updates to include new pathogen-VOC data, which will enrich the classifiers. The sets of VOCs identified potentiate the improvement of the selectivity of non-invasive infection diagnostics using artificial olfaction devices.

Dhadge, VL, Rosa S, Azevedo A, Aires-Barros R, Roque ACA.  2014.  Magnetic Aqueous Two Phase Fishing: An Hybrid Process Technology for Antibody Purification. J. Chromatogr. A. 1339:59-64. AbstractWebsite

The potential to combine aqueous two-phase extraction (ATPE) with magnetic separation was here investigated with the aim of developing a selective non-chromatographic method for the purification of antibodies from cell culture supernatants. Aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG) and dextran were supplemented with several surface modified magnetic particles (MPs) at distinct salt concentrations. The partition of pure human IgG in the upper and lower phases as well as the amount adsorbed at the MPs surface was investigated, indicating that MPs coated with dextran and gum Arabic established the lowest amount of non-specific interactions. The binding capacity of gum arabic coated particles modified with aminophenyl boronic acid (GA-APBA-MP) was were found to be excellent in combination with the ATPS system, yielding high yields of antibody recovery (92%) and purity (98%) from cell culture supernatants. The presence of MPs in the ATPS was found to speed up phase separation (from 40 to 25 min), to consume a lower amount of MPs (half of the amount needed in magnetic fishing) and to increase the yield and purity of a mAb purified from a cell culture supernatant, when compared with ATPE or magnetic fishing processes alone.

Fernandes, CSM, Pina AS, Batalha ÍL, Roque ACA.  2017.  Magnetic fishing of recombinant green fluorescent proteins and tagged proteins with designed synthetic ligands. Separation Science and Technology. AbstractWebsite

Biomimetic ligands have emerged to overcome disadvantages inherent in biological ligands. In particular, the Ugi reaction can generate scaffolds where molecular diversity can be introduced, allowing the synthesis and screening of ligand libraries in a high-throughput manner against a variety of biological targets. Two adsorbents bearing Ugi-based synthetic ligands, coined A4C7 and A7C1, were previously developed for the selective recovery of green fluorescent protein (GFP) and RKRKRK-tagged GFP directly from Escherichia coli crude extracts. This work describes, for the first time, the in situ synthesis of Ugi-based ligands on magnetic beads and their application in the magnetic recovery of cognate proteins.

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.
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.
Borlido, L, Azevedo AM, Roque ACA, Aires-Barros MR.  2013.  Magnetic separations in biotechnology. Biotechnology Advances. 31(8):1374-1385. AbstractWebsite

Magnetic separations are probably one of the most versatile separation processes in biotechnology as they are able to purify cells, viruses, proteins and nucleic acids directly from crude samples. The fast and gentle process in combination with its easy scale-up and automation provide unique advantages over other separation techniques. In the midst of this process are the magnetic adsorbents tailored for the envisioned target and whose complex synthesis spans over multiple fields of science. In this context, this article reviews both the synthesis and tailoring of magnetic adsorbents for bioseparations as well as their ultimate application.

Rebordão, G, Palma SICJ, Roque ACA.  2020.  Microfluidics in Gas Sensing and Artificial Olfaction. Sensors . 20(20):5742. AbstractPDF

Rapid, real-time, and non-invasive identification of volatile organic compounds (VOCs)
and gases is an increasingly relevant field, with applications in areas such as healthcare, agriculture,
or industry. Ideal characteristics of VOC and gas sensing devices used for artificial olfaction include
portability and affordability, low power consumption, fast response, high selectivity, and sensitivity.
Microfluidics meets all these requirements and allows for in situ operation and small sample amounts,
providing many advantages compared to conventional methods using sophisticated apparatus such
as gas chromatography and mass spectrometry. This review covers the work accomplished so far
regarding microfluidic devices for gas sensing and artificial olfaction. Systems utilizing electrical
and optical transduction, as well as several system designs engineered throughout the years are
summarized, and future perspectives in the field are discussed.

Pina, AS, Dias AMGC, Ustok FI, Khoury GE, Fernandes CSM, Branco RJF, Lowe CR, Roque ACA.  2015.  Mild and cost-effective green fluorescent protein purification employing small synthetic ligands. Journal of Chromatography A. 1418:83-93. AbstractWebsite

Abstract The green fluorescent protein (GFP) is a useful indicator in a broad range of applications including cell biology, gene expression and biosensing. However, its full potential is hampered by the lack of a selective, mild and low-cost purification scheme. In order to address this demand, a novel adsorbent was developed as a generic platform for the purification of \{GFP\} or \{GFP\} fusion proteins, giving \{GFP\} a dual function as reporter and purification tag. After screening a solid-phase combinatorial library of small synthetic ligands based on the Ugi-reaction, the lead ligand (A4C7) selectively recovered \{GFP\} with 94% yield and 94% purity under mild conditions and directly from Escherichia coli extracts. Adsorbents containing the ligand \{A4C7\} maintained the selectivity to recover other proteins fused to GFP. The performance of \{A4C7\} adsorbents was compared with two commercially available methods (immunoprecipitation and hydrophobic interaction chromatography), confirming the new adsorbent as a low-cost viable alternative for \{GFP\} purification.

Batalha, IL, Zhou H, Lilley K, Lowe CR, Roque ACA.  2016.  Mimicking nature: Phosphopeptide enrichment using combinatorial libraries of affinity ligands. Journal of Chromatography A. 1457:76–87.: Elsevier B.V. AbstractWebsite

Phosphorylation is a reversible post-translational modification of proteins that controls a plethora of cellular processes and triggers specific physiological responses, for which there is a need to develop tools to characterize phosphorylated targets efficiently. Here, a combinatorial library of triazine-based synthetic ligands comprising 64 small molecules has been rationally designed, synthesized and screened for the enrichment of phosphorylated peptides. The lead candidate (coined A8A3), composed of histidine and phenylalanine mimetic components, showed high binding capacity and selectivity for binding mono- and multi-phosphorylated peptides at pH 3. Ligand A8A3 was coupled onto both cross-linked agarose and magnetic nanoparticles, presenting higher binding capacities (100-fold higher) when immobilized on the magnetic support. The magnetic adsorbent was further screened against a tryptic digest of two phosphorylated proteins ($\alpha$- and $\beta$-caseins) and one non-phosphorylated protein (bovine serum albumin, BSA). The MALDI-TOF mass spectra of the eluted peptides allowed the identification of nine phosphopeptides, comprising both mono- and multi-phosphorylated peptides.