http://hdl.handle.net/10174/14477 2026-04-04T09:12:30Z 2026-04-04T09:12:30Z Eustáquio, Raquel Caldeira, Ana Teresa Caldeira Candeias, António Pereira, António http://hdl.handle.net/10174/40512 2026-01-18T22:15:02Z 2025-12-01T00:00:00Z

Title: Affordable small molecules as promising fluorescent labels for biomolecules: application in RNA-FISH probes Authors: Eustáquio, Raquel; Caldeira, Ana Teresa Caldeira; Candeias, António; Pereira, António Abstract: Fluorescent labels play a vital role in a wide range of contemporary scientific techniques, including direct and indirect immunochemistry, fluorescence microscopy, histochemistry, flow cytometry, and fluorescence in situ hybridization (FISH). Small fluorescent labels provide substantial practical advantages by allowing multiple fluorophores to be conjugated to a single biomolecule, thereby enhancing fluorescence signal optimization. Widely used fluorescent labels are often too costly for routine applications and typically exhibit small Stokes shifts, limiting their practical utility. In this study, we report three novel small molecules as promising fluorescent labels for biomolecules, synthesized via an efficient, straightforward, and cost-effective approach. Additionally, we assess the fluorescence properties of the three new small molecules as potential fluorescent labels for biomolecules. Six novel fluorescent oligonucleotide probes were synthesized, including three targeting the rRNA region of eukaryotic cells (EUK516) and three targeting the rRNA region of prokaryotic cells (EUB338). The synthesized fluorescent probes were evaluated using microorganisms from the culture collection of the Laboratory of Biodegradation and Biotechnology at the HERCULES Laboratory, University of Évora. The probes demonstrated effective performance in RNA-FISH applications, highlighting the potential of these new small molecules for biomolecule labeling and bioimaging.

2025-12-01T00:00:00Z Eustáquio, Raquel Ramalho, João Paulo Caldeira, Ana Teresa Pereira, António http://hdl.handle.net/10174/39229 2025-09-04T14:09:24Z 2024-01-22T00:00:00Z

Title: Inexpensive small molecules as promising fluorescent labels for biomolecules. Authors: Eustáquio, Raquel; Ramalho, João Paulo; Caldeira, Ana Teresa; Pereira, António Abstract: Fluorescent labels are indispensable in various modern scientific applications, including direct and indirect immunochemistry, fluorescence microscopy, histochemistry, flow cytometry and fluorescence in situ hybridization (FISH) [1,2]. Small fluorescent labels offer significant practical benefits, enabling the optimization of fluorescence signals through the attachment of multiple fluorophores to a single biomolecule [3,4]. The commonly used fluorescent labels are prohibitively expensive for regular use in routine applications and most of them have small Stokes shifts. In this work we present three new small molecules (Figure 1), as promising fluorescent labels for biomolecules, obtained through an efficient, straightforward, and cost-effective synthetic strategy. Additionally, we evaluate the fluorescent three new small molecules labels as potentially effective fluorescent labels for biomolecules. Six new fluorescent oligonucleotide probes have been obtained, three directed to the rRNA region of eukaryotic cells (EUK516) and three to the rRNA region of prokaryotic cells (EUB338). The developed fluorescent probes were tested on microorganisms belonging to the culture collection of the Laboratory of Biodegradation and Biotechnology of the HERCULES Laboratory (University of Évora), showing effective performance as RNA-FISH probes. Density functional theory and time-dependent density functional theory calculations were carried out to gain insights into the observed photophysical properties. These findings evidenced the applicability of these new small molecules in labeling of biomolecules and bioimaging.

2024-01-22T00:00:00Z Eustáquio, Raquel Caldeira, Ana Teresa Arantes, Sílvia Candeias, António Pereira, António http://hdl.handle.net/10174/39227 2025-09-04T14:09:11Z 2025-01-22T00:00:00Z

Title: Synthesis of new inexpensive fluorescent water-soluble amine-reactive reagents for biomolecules labeling: application to cultural heritage. Authors: Eustáquio, Raquel; Caldeira, Ana Teresa; Arantes, Sílvia; Candeias, António; Pereira, António Abstract: Cultural heritage assets are being continuously submitted to physical, chemical and biological degradation. Biodegradation is the major factor for their deterioration, with microorganisms being of critical importance due to their metabolism and proliferation. In this context, it is frequently necessary identify and locate individual microorganisms and detect small amounts of biomolecules, as proteins, to know, evaluate and understand the biodeterioration processes for the most appropriate restoration procedures or conservation conditions. Fluorescent labeling is one of the most widely used techniques for bioanalytical applications, with fluorescence-based detection drawing growing interest in both the advancement of fluorescence instrumentation and the design of new fluorophores. Fluorescent labels, commonly employed in highly sensitive analytical techniques to detect and track biomolecules in vital fields like cellular biology, medicine, medicinal chemistry, and environmental science, remain prohibitively expensive for routine use in standard applications. In this work, using 4-diethylaminobenzaldehyde as starting material, we developed a simple, cost-effective and efficient synthetic strategy to produce new inexpensive small molecules as effective fluorescent labels for biomolecules present in cultural heritage. This work will enable the advancement of new methodologies for diagnosing biodegradation, thereby contributing to the development of preventive strategies and effective remediation in the field of cultural heritage.

2025-01-22T00:00:00Z Eustáquio, Raquel Caldeira, Ana Teresa Arantes, Sílvia Candeias, António Pereira, António http://hdl.handle.net/10174/39224 2025-09-04T14:08:50Z 2023-12-07T00:00:00Z

Title: New 4-styrylcoumarin derivatives as potentials fluorescent labels for biomolecules: application in RNA-FISH probes. Authors: Eustáquio, Raquel; Caldeira, Ana Teresa; Arantes, Sílvia; Candeias, António; Pereira, António Abstract: Currently, fluorescence microscopy is one of the highly sensitive imaging techniques that allow visualize, detect, and track biomolecules in analytical studies in many important scientific areas as cellular biology, environmental sciences, medicine, pharmacy, among others. One of the great advantages of fluorescence microscopy is the possibility to use several fluorescent labels to detect different biomolecules and produce multicolour images allowing the identification, in vitro and in vivo, of specific components of complex biomolecular assemblies, as well analysis of their interactions. Fluorescent labels can produce chemically stable and small bioconjugates, with insignificant interference on the structure and biological functions of the unlabelled biomolecules. The amine-reactive fluorescent labels, due to the abundance of amino groups or its easy insertion into biomolecules, are the most frequently used to prepare bioconjugates for a multiplicity of biological applications as fluorescence in situ hybridization (FISH), histochemistry, cell tracing, receptor binding, or direct and indirect immunochemistry. Presently, the most widely used available fluorescent labels are very expensive and that's why coumarin derivatives can be a solution to develop low-cost new brightness fluorophores. In this work, we developed a low cost and effective synthetic strategy to synthesize six new 4-styrylcoumarin derivatives (Figure 1), as potentials fluorescent labels for biomolecules, using the inexpensive 7-diethylamino-4-methylcoumarin as a starting material. New fluorescent oligonucleotide probes have been obtained, six directed to the rRNA region of eukaryotic cells (EUK516) and six to the rRNA region of prokaryotic cells (EUB338). The developed fluorescent probes were tested on microorganisms belonging to the culture collection of the Laboratory of Biodegradation and Biotechnology of the HERCULES Laboratory (University of Évora), namely Saccharomyces cerevisiae and Bacillus showing effective performance as RNA-FISH probes. These findings evidenced the applicability of the new 4-styrylcoumarin derivatives in labeling of biomolecules and bioimaging.

2023-12-07T00:00:00Z