ABSTRACT
In the present research, Fe-based metal-organic frameworks (MIL-101(Fe)-NH2) nanoparticles were synthesized by simple solvothermal methods and used to assay Cr(â ¥). The MIL-101(Fe)-NH2 performs dual functions: the 2-aminoterephthalic acid (NH2-BDC) ligand endows a strong fluorescence emission, and the Fe metal nodes are able to facilitate the oxidation of 3,3',5,5'- tetramethylbenzidine (TMB) directly, resulting in the generation of oxidized-TMB (ox-TMB). Our research results showed that reducing agents such as ascorbic acid (AA) can collapse the structures of MIL-101(Fe)-NH2 because of the reduction of Fe3+ by AA, resulting in release of NH2-BDC. In the presence of Cr(â ¥), the fluorescence intensity of the MIL-101(Fe)-NH2 + AA system will be decreased due to the competitive reduction of Fe3+ and Cr(â ¥). Nevertheless, Cr(â ¥) can significantly accelerate the oxidation of TMB by MIL-101(Fe)-NH2 as it boosts the electron transfer rate between Fe3+ and Fe2+. Therefore, a fluorescent/colorimetric dual-mode platform was developed for the detection of Cr(â ¥) with an extensive linear range (7.5-750 µg/L and 13.3-1000 µg/L) as well as a remarkably low detection limit (0.99 µg/L and 1.98 µg/L). This MOF with the ability to release ligands not only provides inspiration for the design of new luminescent materials, but also offers a novel and reliable solution for the detection of Cr(â ¥).
ABSTRACT
Beta-amyloid (Aß), the most pivotal pathological hallmark for Alzheimer's disease (AD) diagnosis and drug evaluation, was recognized by TZ095, a high-affinity fluorescent probe developed by rational molecular design. With a TICT mechanism, TZ095 exhibited remarkable affinity with Aß aggregates (Kd = 81.54 nM for oligomers; Kd = 66.70 nM for fibril) and substantial fluorescence enhancement (F/F0 = 44), enabling real-time monitoring of Aß in live cells and nematodes. Significantly, this work used TZ095 to construct a new protocol that can quickly and conveniently monitor Aß changes at the cellular and nematode levels to evaluate the anti-AD efficacy of candidate compounds, and four reported Aß-lowering drug candidates were administrated for validation. Imaging data demonstrated that TZ095 can visually and quantitatively track the effect of Aß elimination after drug treatment. Furthermore, TZ095 excelled in ex vivo histological staining of 12-month-old APP/PS1 mouse brains, accurately visualizing Aß plaques. Integrating CUBIC technology, TZ095 facilitated whole-brain, 3D imaging of Aß distribution in APP/PS1 mice, enabling high-resolution in situ analysis of Aß plaques. Collectively, these innovative applications of TZ095 offer a promising strategy for rapid, convenient, and real-time monitoring of Aß levels in preclinical therapeutic assessments.
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Hydrazine is volatile and highly toxic, causing severe harm to water, soil, air, and organisms. Therefore, real-time detection and long-term monitoring of hydrazine are crucial for environmental protection and human health. Herein, an "OFF-ON" fluorescent probe 5-((10-ethyl-2-methoxy-10 H-phenothiazin-3-yl)methylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (MPD) for hydrazine detection through a nucleophilic addition reaction was developed. MPD could exclusively identify hydrazine through colorimetric and fluorescent dual-channel responses within 30 s, which also demonstrated high sensitivity (detection limit, 12 nM) and a wide pH range (6 -12). The sensing mechanism of MPD was confirmed using theoretical calculations, where fluorescence was emitted following the recognition of hydrazine because of the disappearance of the photoinduced electron transfer (PET) process. Using a smartphone, MPD enabled the quantitative detection of hydrazine in real water samples and sandy soil. Notably, in the process of detecting hydrazine in actual water samples, the establishment of analytical methods and the completion of rapid quantitative detection only required a smartphone and built-in apps. Additionally, we showed that MPD could recognize hydrazine in various environmental samples, including plants, food, hydrazine vapors, and cells. We believe that the fluorescent probe MPD developed in this study and the established smartphone visualization platform will provide a convenient and effective tool for detecting hydrazine in environmental monitoring, food safety assessment, biological system safety, and other fields.
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Carbon dots (CQD) have received significant attention as a novel ratiometric fluorescent pH nanoprobe, owing to their favorable optical properties and excellent biocompatibility. Despite their appealing features, the precise mechanism behind the pH-sensitive photoluminescence of CQDs remains to be fully understood. This study endeavors to unravel the mechanism underlying the pH-responsive ratiometric fluorescence in dual-emission CQDs, synthesized through a one-step hydrothermal method using o-phenylenediamine and oxalic acid as precursors. The resultant CQDs exhibit inherent dual-emission at wavelengths of 383 nm and 566 nm, with the ratiometric fluorescence response tailored by the ratio of precursors, providing a robust tool for pH sensing across a range of 2 to 6. Detailed characterizations, including chemical, morphological, and optical analyses, alongside theoretical insights from time-dependent density functional theory (TD-DFT), elucidate the mechanism underlying the pH-dependent luminescence, attributed to the electron cloud transmission between amide and adjacent carboxyl groups on the CQD surface. The superior performance of these CQDs in real-time pH monitoring is demonstrated through their application in glucose oxidase-catalyzed reactions, showcasing their potential as efficient, reliable nanoprobes for biomedical research and diagnostic applications.
ABSTRACT
BACKGROUND: In recent years, environmental pollution has been increasing due to the excessive emission of toxic ions, which has caused serious harm to human health and ecological environment. There are various methods for detecting Cu2+, S2- and Zn2+, but the traditional ion detection methods have obvious disadvantages, such as poor selectivity and long detection time. Therefore, it is still crucial to develop simple, efficient and rapid detection methods. RESULTS: A fluorescent probe based on benzothiazole, (E)-N'-(3-(benzo[d]thiazol-2-yl)-2-hydroxy-5-methylbenzylidene)-3,4,5-tris(benzyloxy)benzohydrazide (BT), was designed and synthesized. It was characterized using ESI-MS, 1H NMR, and 13C NMR. BT can be used as a chemosensor to detect Cu2+, S2- and Zn2+ in CH3CN/H2O (7:3, v/v, pH = 7.4, HEPES buffer: 0.1 M), with detection limits of 0.301 µM, 0.017 µM, and 0.535 µM, respectively. At an excitation wavelength of 320 nm, BT exhibits an "on-off-on" response to Cu2+/S2- and enhanced fluorescence response to Zn2+, with a change in fluorescence color from orange to green. The coordination ratio of ions to the probe was determined to be 1:1 through Job's plot and hydrogen spectral titration. The recognition mechanism was discussed in conjunction with theoretical calculations. Furthermore, the probe has been successfully used in test strips and medical swabs colorimetry, as well as live cell imaging. SIGNIFICANCE: The probe BT lays the foundation for the design and synthesis of multifunctional fluorescent probes. As a portable detection method, probe BT was used to detect Cu2+, S2- and Zn2+ on strips. Furthermore, the probe was applied to biological cells to detect target ions with low cytotoxicity and excellent cell permeability. This indicating that it can be used as a potential candidate for tracking Cu2+ and S2- in clinical diagnostics and biological systems.
Subject(s)
Benzothiazoles , Copper , Fluorescent Dyes , Zinc , Fluorescent Dyes/chemistry , Fluorescent Dyes/chemical synthesis , Benzothiazoles/chemistry , Copper/chemistry , Copper/analysis , Zinc/chemistry , Zinc/analysis , Humans , Optical Imaging , Spectrometry, Fluorescence , HeLa Cells , Molecular StructureABSTRACT
Ion channels represent a druggable family of transmembrane pore-forming proteins with important (patho)physiological functions. While electrophysiological measurement (manual patch clamp) remains the only direct method for detection of ion currents, it is a labor-intensive technique. Although automated patch clamp instruments have become available to date, their high costs limit their use to large pharma companies or commercial screening facilities. Therefore, fluorescence-based assays are particularly important for initial screening of compound libraries. Despite their numerous disadvantages, they are highly amenable to high-throughput screening and in many cases, no sophisticated instrumentation or materials are required. These features predispose them for implementation in early phases of drug discovery pipelines (hit identification), even in an academic environment. This review summarizes the advantages and pitfalls of individual methodological approaches for identification of ion channel modulators employing fluorescent probes (i.e., membrane potential and ion flux assays) with emphasis on practical aspects of their adaptation to high-throughput format.
ABSTRACT
Cancer is anticipated to become the pioneer reason of disease-related deaths worldwide in the next two decades, underscoring the urgent need for personalized and adaptive treatment strategies. These strategies are crucial due to the high variability in drug efficacy and the tendency of cancer cells to develop resistance. This study investigates the potential of theranostic nanotechnology using three innovative fluorescent polymers (FP-1, FP-2, and FP-3) encapsulated in niosomal carriers, combining therapy (chemotherapy and radiotherapy) with fluorescence imaging. These cargoes are assessed for their cytotoxic effects across three cancer cell lines (A549, MCF-7, and HOb), with further analysis to determine their capacity to augment the effects of radiotherapy using a Linear Accelerator (LINAC) at specific doses. Fluorescence microscopy is utilized to verify their uptake and localization in cancerous versus healthy cell lines. The results confirmed that these niosomal cargoes not only improved the antiproliferative effects of radiotherapy but also demonstrate the practical application of fluorescent polymers in in vitro imaging. This dual function underscores the importance of dose optimization to maximize therapeutic benefits while minimizing adverse effects, thereby enhancing the overall efficacy of cancer treatments.
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Chiral amino acids (AAs) are essential in metabolism and understanding physiological processes, and they could be used as biomarkers for the diagnosis of different diseases. In this study, chiral Cdots@Van were prepared by postmodifying an achiral Cdots core with vancomycin for recognizing and determining the enantiomeric excess (ee) of tyrosine (Tyr) enantiomers. The fluorescence response of Cdots@Van is based on an "on-off" strategy, with different quenching percentages for d- and l-tyrosine. Interestingly, the circular dichroism (CD) spectrum of Cdots@Van responded to only one form of Tyr enantiomer, specifically d-Tyr, and remained nearly unchanged upon the addition of l-Tyr. Quantum mechanical (QM) calculations were in excellent agreement with the experimental results, confirming the stronger binding affinity of Cdots@Van for d-Tyr compared to l-Tyr. We further investigated the chiral recognition ability of the interconnected vancomycin particles, which was synthesized using the EDC/NHS coupling reaction between vancomycin molecules without a Cdots core. Surprisingly, unlike free vancomycin molecules, interconnected vancomycin displayed an enantiomeric recognition ability by CD spectroscopy, similar to what was observed for Cdots@Van. Crucially, this chiral probe has been successfully utilized for cell imaging applications.
ABSTRACT
Crystal polymorphism serves as a strategy to study the conformational flexibility of proteins. However, the relationship between protein crystal packing and protein conformation often remains elusive. In this study, two distinct crystal forms of a green fluorescent protein variant, NowGFP, are compared: a previously identified monoclinic form (space group C2) and a newly discovered orthorhombic form (space group P212121). Comparative analysis reveals that both crystal forms exhibit nearly identical linear assemblies of NowGFP molecules interconnected through similar crystal contacts. However, a notable difference lies in the stacking of these assemblies: parallel in the monoclinic form and perpendicular in the orthorhombic form. This distinct mode of stacking leads to different crystal contacts and induces structural alteration in one of the two molecules within the asymmetric unit of the orthorhombic crystal form. This new conformational state captured by orthorhombic crystal packing exhibits two unique features: a conformational shift of the ß-barrel scaffold and a restriction of pH-dependent shifts of the key residue Lys61, which is crucial for the pH-dependent spectral shift of this protein. These findings demonstrate a clear connection between crystal packing and alternative conformational states of proteins, providing insights into how structural variations influence the function of fluorescent proteins.
ABSTRACT
Exocytosis is a dynamic physiological process that enables the release of biomolecules to the surrounding environment via the fusion of membrane compartments to the plasma membrane. Understanding its mechanisms is crucial, as defects can compromise essential biological functions. The development of pH-sensitive optical reporters alongside fluorescence microscopy enables the assessment of individual vesicle exocytosis events at the cellular level. Manual annotation represents, however, a time-consuming task, prone to selection biases and human operational errors. Here, we introduce ExoJ, an automated plugin based on ImageJ2/Fiji. ExoJ identifies user-defined genuine populations of exocytosis events, recording quantitative features including intensity, apparent size and duration. We designed ExoJ to be fully user-configurable, making it suitable to study distinct forms of vesicle exocytosis regardless of the imaging quality. Our plugin demonstrates its capabilities by showcasing distinct exocytic dynamics among tetraspanins and vesicular SNAREs protein reporters. Assessment of performance on synthetic data showed ExoJ is a robust tool, capable to correctly identify exocytosis events independently of signal-to-noise ratio conditions. We propose ExoJ as a standard solution for future comparative and quantitative studies of exocytosis.
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Epigenetic modifications (methylation, acetylation, etc.) of core histones play a key role in regulation of gene expression. Thus, the epigenome changes strongly during various biological processes such as cell differentiation and dedifferentiation. Classical methods of analysis of epigenetic modifications such as mass-spectrometry and chromatin immuno-precipitation, work with fixed cells only. Here we present a genetically encoded fluorescent probe, MPP8-Green, for detecting H3K9me3, a histone modification associated with inactive chromatin. This probe, based on the chromodomain of MPP8, allows for visualization of H3K9me3 epigenetic landscapes in single living cells. We used this probe to track changes in H3K9me3 landscapes during the differentiation of induced pluripotent stem cells (iPSCs) into induced neurons. Our findings revealed two major waves of global H3K9me3 reorganization during 4-day differentiation, namely on the first and third days, whereas nearly no changes occurred on the second and fourth days. The proposed method LiveMIEL (Live-cell Microscopic Imaging of Epigenetic Landscapes), which combines genetically encoded epigenetic probes and machine learning approaches, enables classification of multiparametric epigenetic signatures of single cells during stem cell differentiation and potentially in other biological models.
Subject(s)
Cell Differentiation , Epigenesis, Genetic , Fluorescent Dyes , Histones , Induced Pluripotent Stem Cells , Cell Differentiation/genetics , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/cytology , Histones/metabolism , Histones/genetics , Humans , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Neurons/metabolism , Neurons/cytology , Animals , MiceABSTRACT
Circularly polarized luminescence (CPL) from chrial molecules is attracting much attention due to its potential in optical materials. However, formulation of CPL emitters as molecular solids typically deteriorates photophysical properties in the aggregated state leading to quenching and unpredictable changes in CPL behavior impeding materials development. To circumvent these shortcomings, a supramolecular approach can be used to isolate cationic dyes in a lattice of cyanostar-anion complexes that suppress aggregation-caused quenching and which we hypothesize can preserve chiroptical properties. Herein, we verify for the first time that supramolecular assembly of small-molecule, ionic isolation lattices (SMILES), allows translation of molecular ECD and CPL properties to solids. A series of cationic helicenes that display increasing chiroptical response, is investigated. Crystal structures of three different packing motifs all show spatial isolation of dyes by the anion complexes. We observe the photophysical and chiroptical properties of all helicenes are seamlessly translated to water soluble nanoparticles by the SMILES method. Also, a DMQA helicene is used as FRET acceptor in SMILES nanoparticles of intensely absorbing rhodamine antennae to generate an 18-fold boost in CPL brightness. These features offer promise for reliably accessing bright materials with programmable CPL properties.
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Fluorescent dye films on transparent substrates are essential for OLEDs, flexible displays, X-ray detection, and wireless optical communications. However, their efficiency is often hampered by fluorescence trapping due to total internal reflection (TIR) and waveguiding. This study tackles this longstanding challenge by reconceptualizing the integration of dye films with nanoantenna metasurfaces. Traditional methods involve directly spin-coating films onto c-Si metasurfaces on quartz substrates, resulting in edge luminescence and weak inner signals. We present a straightforward, adjustable approach by integrating dye films on the opposite side of quartz substrates, reaching a 2.5-fold photoluminescence enhancement and improving the uniformity of the emission compared to the conventional methods. These gains stem from redirecting a significant portion of leaked fluorescence light trapped inside the substrate into free space, surpassing TIR conditions through in-plane diffraction orders of the metasurfaces across the full RGB spectrum. Our findings facilitate the design of more efficient luminescent devices.
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The extensive use of fluoride in agriculture, industry, medicine, and daily necessities has raised growing concerns about fluoride residue. To date, real-time visual detection and efficient removal of fluoride ions from water remain greatly desirable. Herein, nano-CAU-10-NH2@RhB is introduced as a ratiometric fluorescent probe and efficient scavenger for the intelligent detection and removal of fluoride ions. CAU-10-NH2@RhB is readily obtained through one-pot synthesis and exhibits high sensitivity and selectivity for real-time fluoride ion detection, with a naked-eye distinguishable color change from pink to blue. A portable device for point-of-care testing was developed based on color hue analysis readout using a smartphone. A quantitative response was achieved across a wide concentration range, with a detection limit of 54.2 nM. Adsorption experiments suggest that nano-CAU-10-NH2@RhB serves as an efficient fluoride ion scavenger, with a fluoride adsorption capacity of 49.3 mg/g. Moreover, the mechanistic study revealed that hydrogen bonds formed between fluoride ions and amino groups of CAU-10-NH2@RhB are crucial for the detection and adsorption of fluoride ions. This analysis platform was also used for point-of-care quantitative visual detection of fluoride ions in food, water, and toothpaste.
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Glycoproteins perform vital functions in numerous biological processes and have important clinical implications. Many glycoproteins have been used as biomarkers and therapeutic targets for disease diagnosis. Due to low concentration of glycoprotein biomarkers and the presence of high-abundance interfering species in biological samples, a selective and sensitive detection method for glycoprotein is essential for real-world applications. In this study, we develop an oriented surface imprinted microplate-based fluorescent biosensor by boronate-affinity sandwich assay (BASA) for the specific, sensitive and high throughput determination of glycoproteins in complex samples. The structure of the BASA is based on sandwich formation between boronate affinity-oriented surface-imprinted microplates, target glycoproteins, and boronate affinity fluorescence probes. The imprinted microplates ensure the high specificity, high affinity and high throughput, while the fluorescence probes, consisting of boronic acid-modified CdTe QDs, provide high sensitivity. The proposed approach could exhibit a wide linear range of 1â¯ng/mL-105â¯ng/mL, with a low LOD of 0.528â¯ng/mL using horseradish peroxidase (HRP) as a model glycoprotein. As compared with traditional "turn off" fluorescent sensor, the developed "turn on" fluorescent sensor provided three orders of magnitude higher sensitivity at least. The fluorescent biosensor achieved average recoveries ranging from 96.8â¯% to 106.0â¯% in urine samples.
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Accurate interpretation of in vivo wide-field fluorescent imaging (WFFI) data requires precise separation of raw fluorescence signals into neural and hemodynamic components. The classical Beer-Lambert law-based approach, which uses concurrent 530-nm illumination to estimate relative changes in cerebral blood volume (CBV), fails to account for the scattering and reflection of 530-nm photons from non-neuronal components leading to biased estimates of CBV changes and subsequent misrepresentation of neural activity. This study introduces a novel linear regression approach designed to overcome this limitation. This correction provides a more reliable representation of CBV changes and neural activity in fluorescence data. Our method is validated across multiple datasets, demonstrating its superiority over the classical approach.
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Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), continues to pose a significant global health threat. Identifying new druggable targets is crucial for the advancement of drug development. Equally critical is the development of precise methods for monitoring Mtb to effectively combat this disease. Addressing these needs, our study pinpointed the pore domain (PD) of MtbMmpL3 as a new binding site for virtual screening, which led to the discovery of the small molecule ZY27. To confirm the binding site and action mode of ZY27, we employed cosolvent molecular dynamics (CMD), steered molecular dynamics (SMD), and long timescale molecular dynamics (MD) simulations of 5⯵s. These in silico studies verified that ZY27 binds to the PD of MtbMmpL3. In antimicrobial activity tests, ZY27 exhibited potent anti-Mtb activity and high selectivity among mycobacterial species. Whole-genome sequencing of spontaneous ZY27-resistant Mtb variants, complemented by acid-fast staining experiments, confirmed that ZY27 specifically targets MtbMmpL3. Utilizing the ligand-protein binding data, we designed and synthesized two solvatochromic fluorescent probes, 27FP1 and 27FP2, based on ZY27. Further investigations through flow cytometry and confocal microscopy confirmed that these probes specifically label Mtb cells via the MtbMmpL3 binding mechanism.
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Melting of brain sphingomyelin (bSM) manifests as a broad feature in the DSC curve that encompasses the temperature range of 25 - 45 °C, with two distinguished maxima originating from the phase transitions of two the most abundant components: C24:1 (Tm,1) and C18:0 (Tm,2). While C24:1/C18:0 sphingomyelin transforms from the gel/ripple phase to the fluid/fluid phase, the dynamics of water molecules in the interfacial layer remain completely unknown. Therefore, we carried out a calorimetric (DSC), spectroscopic (temperature-dependent UV-Vis and fluorescence) and MD simulation study of bSM in the absence/presence of Laurdan® (bSM ± L) suspended in Britton-Robinson buffer with three different pH values, 4 (BRB4), 7 (BRB7) and 9 (BRB9), and of comparable ionic strength (I = 100mM). According to DSC, TÌ m, 1 (≈ 34.5 °C/≈ 32.1 °C) and TÌ m, 2 (≈ 38.0 °C/≈ 37.2 °C) of bSM suspended in BRB4, BRB7, and BRB9 in the absence/presence of Laurdan® are found to be practically pH-independent. Turbidity-based data (UV-Vis) detected both qualitative and quantitative differences in the response of bSM suspended in BRB4/BRB7/BRB9 (TÌ m: â¼ 35 °C/32.0 ± 0.2 °C/36.4 ± 0.4), suggesting an intricate interplay of weakening of van der Waals forces between their hydrocarbon chains and of increased hydration in the polar headgroups region during melting. The temperature-dependent response of Laurdan® reported a discontinuous, pH-dependent change in the reorientation of interfacial water molecules that coincides with the melting of C24:1 lipids (on average, TÌ m (LTC/HTC): ≈ 31.8 °C/30.6 °C/30.5 °C). MD simulations elucidated the impact of Laurdan® on a change in the physicochemical properties of bSM lipids and characterized the hydrogen bond network at the interface at 20 °C and 50 °C.
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In this study, a novel fluorescent probe, MAC-2, for the detection of Au3+ was designed and synthesised using cellulose as a carrier combined with benzothiazole derivatives. The structure of the probe was confirmed by SEM, XRD, FTIR, and 1H NMR, also the optical properties of the product were investigated. MAC-2 showed bright green fluorescence under a 365 nm UV lamp and exhibited significant quenching behaviour toward Au3+. MAC-2 utilises more sustainable biomass resources, featuring green and biodegradable characteristics that meet environmental requirements. Compared with most reported probes, it exhibits notable fluorescence properties. The limit of detection (LOD) is as low as 0.057 µM, and the response time is 1 min. It also demonstrates good specific recognition and anti-interference abilities. In addition, a smartphone was used as a portable signal processing device to achieve rapid detection of Au3+ concentration. Meanwhile, MAC-2 was successfully prepared as a fluorescent test strip, providing a potential application for the convenient detection of Au3+. The high sensitivity and selectivity exhibited by cellulose-based fluorescent probes in detecting Au3+ offer valuable insights and new ideas for the detection of other metal ions and biomolecules. These inspirations will help promote the continuous development of research and applications in related fields.
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Archaeological pottery are the most numerous objects found during excavations and reflect the culinary practices of the past. However, their functionality for cooking/storing specific foods or drinks cannot be deduced solely from comparing their shapes and sizes. Analysis of protein residues bound to ceramics can reveal the protein/animal type through their amino acid sequence, thus enabling direct identification of food types. Therefore, the aim of our experimental study was to test sixteen aptamers for the analysis of proteinaceous organic residues found within the porous structure of pottery. Traditionally prepared archaeological ceramic replicas were cooked for 5 days in various food/protein suspensions, were UV aged, buried for a year, excavated, and extensively cleaned. Their shards were analysed using immunofluorescence microscopy with aptamers. Results show that eight aptamers (Clone1 and Kirby for egg residuals; seqU5 and BLG14 for milk residuals; HA for blood residuals; Gli4 for gluten residuals; Par1 for fish residuals; and D1 for collagen residuals) produced a successful/specific immunofluorescence microscopy result when they were hybridised to shards containing target protein residuals. Interestingly, on whole egg control samples, when the egg lysozyme-targeting aptamer Kirby was used, fluorescence intensity was 3.1 times greater compared to that observed with anti-ovalbumin antibodies.