Self-Assembly Multivalent Fluorescence-Nanobody Coupled Multifunctional Nanomaterial with Colorimetric Fluorescence and Photothermal to Enhance Immunochromatographic Assay

ACS Nano, Journal Year: 2023, Volume and Issue: 17(19), P. 19359 - 19371

Published: Oct. 2, 2023

The multimodal lateral flow immunoassay (LFIA) has provided accurate and reliable results for fast immediate detection. Nonetheless, LFIA remains challenging to develop biosensors with high sensitivity tolerance matrix interference in agro-food. In this study, we developed a self-assembled multivalent fluorescence-nanobody (Nb26-EGFP-H6) 16.5-fold 30-fold higher affinity than monovalent nanobody (Nb26). Based on the Nb26-EGFP-H6, synthesized enhanced immune-probes Zn-CN@Nb26-EGFP-H6 by pyrolyzing oxidizing an imidazolating zeolite framework-8 (ZIF-8) obtain photothermal metal–carbon nanomaterials (Zn-CN) immobilizing Nb26-EGFP-H6. rough porous structure of Zn-CN large surface area facilitates enrichment immobilization antibodies. A trimodal (tLFIA) colorimetric, fluorescent, triple signal outputs was constructed detection aflatoxin B1 (AFB1) maize. Attractively, Zn-CN-based tLFIA's multiplex guarantees sensitive AFB1, limits 0.0012 ng/mL (colorimetric signals), 0.0094 (fluorescent 0.252 (photothermal signals). trimode immunosensor 628-fold 42-fold that original Nb26-based ELISA (IC50) unimodal (LOD). This work provides idea constructing sensitive, tolerant efficient analytical platform rapidly detecting AFB1 food.

Language: Английский

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Engineering paralog-specific PSD-95 recombinant binders as minimally interfering multimodal probes for advanced imaging techniques

eLife, Journal Year: 2024, Volume and Issue: 13

Published: Jan. 3, 2024

Despite the constant advances in fluorescence imaging techniques, monitoring endogenous proteins still constitutes a major challenge particular when considering dynamics studies or super-resolution imaging. We have recently evolved specific protein-based binders for PSD-95, main postsynaptic scaffold at excitatory synapses. Since synthetic recombinant recognize epitopes not directly involved target protein activity, we consider them here as tools to develop PSD-95 probes. After confirming their lack of impact on function, validated use intrabody fluorescent further engineered probes and demonstrated usefulness different modalities (STED, PALM, DNA-PAINT) both live fixed neurons. Finally, exploited enrich synapse genetically encoded calcium reporters. Overall, demonstrate that these constitute robust efficient platform selectively monitor using various techniques.

Language: Английский

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Advancing cell biology with nanoscale fluorescence imaging: essential practical considerations

Trends in Cell Biology, Journal Year: 2024, Volume and Issue: 34(8), P. 671 - 684

Published: Jan. 5, 2024

Here, we provide an overview of the key factors to consider when using fluorescence nanoscopy (FN), including biological questions that can be addressed and aspects might improve reliability effectiveness FN experiments.We cover main related sample preparation, selection appropriate fixation, affinity-based labels, fluorescent dyes.We discuss current limitations possible future developments in field would facilitate a broader application FN.We multiplexing possibilities (allowing simultaneous detection multiple targets single experiment), live cell imaging for study cellular molecular dynamic processes, quantitative workflows. Recently, biologists have gained access several far-field (FN) technologies allow observation components with ~20 nm resolution. is revolutionizing biology by enabling visualization previously inaccessible subcellular details. While technological advances microscopy are critical field, optimal preparation labeling equally important often overlooked experiments. In this review, methodological experimental must considered performing FN. We present concepts dyes, multiplexing, approaches, microscopy. Consideration these greatly enhances FN, making it exquisite tool numerous applications. Conventional microscopy, widefield confocal has been essential studying morphology composition various organelles localization molecules. The resolution (see Glossary) techniques, usually above ~200 nm, limit macromolecular arrangements nanoscale structures. processes (<20 nm) major step forward because bridges world macromolecules. This effort facilitated recent decades use electron (EM), which provides morphological information at level [1.Winey M. et al.Conventional transmission microscopy.Mol. Biol. Cell. 2014; 25: 319-323Crossref PubMed Google Scholar] expenses limited identifications lack applicability. These overcome achieved resolving capabilities progressively reaching those attained EM. Several approaches based on enable researchers address <20 were difficult answer classical Paradigmatic examples applications include characterization periodicity actin rings synaptic sites neurons, structure nuclear pore complexes, maturation viral particles, organization mitochondrial cristae, mechanisms apoptosis, functioning signaling pathways [2.Fornasiero E.F. Opazo F. Super-resolution biologists.BioEssays. 2015; 37: 436-451Crossref Scholar]. Recent studies reached extremely high resolutions [3.Reinhardt S.C.M. al.Ångström-resolution microscopy.Nature. 2023; 617: 711-716Crossref Scopus (21) even enabled follow events such as stepping behavior kinesin vitro cells [4.Wolff J.O. al.MINFLUX dissects unimpeded walking kinesin-1.Science. 379: 1004-1010Crossref (20) Scholar,5.Deguchi T. al.Direct motor protein living MINFLUX.Science. 1010-1015Crossref (22) render individual molecules, GABA receptors, detail almost comparable cryo-EM [6.Shaib A.H. al.Visualizing proteins expansion microscopy.bioRxiv. (Published online March 10, 2023. https://doi.org/10.1101/2022.08.03.502284)Google compendium summarizes practical keep mind harnessing power nanoscopic biology. By bridging theory practice, roadmap researchers, equipping them know-how successfully navigate intricacies implementing, executing, deriving meaningful data from design experiments starts most suitable technique, each its own specific strengths (Figure 1A ; reviews different see [7.Jacquemet G. al.The biologist's guide super-resolution microscopy.J. Cell Sci. 2020; 133jcs240713Crossref (32) Scholar,8.Bond C. al.Technological processes.Mol. 2022; 82: 315-332Abstract Full Text PDF (25) Scholar]). Two strategies currently able reliably <20-nm samples: camera-based single-molecule (SMLM) [9.Rust M.J. al.Sub-diffraction-limit stochastic optical reconstruction (STORM).Nat. Methods. 2006; 3: 793-796Crossref (6072) minimal photon fluxes (MINFLUX) [10.Balzarotti al.Nanometer tracking molecules fluxes.Science. 2017; 355: 606-612Crossref (657) Based utilized perform on–off switching fluorophores required obtain super-resolved image, SMLM names; example, (STORM), photoactivated (PALM), DNA-points accumulation topography (DNA-PAINT) [11.Jungmann R. al.Single-molecule kinetics transient binding DNA origami.Nano Lett. 2010; 10: 4756-4761Crossref (576) addition two strategies, depending exact settings, stimulated emission depletion (STED) [12.Hell S.W. Wichmann J. Breaking diffraction emission: stimulated-emission-depletion microscopy.Opt. 1994; 19: 780Crossref achieve ~50 below, although laser not compatible conventional samples. technologies, approach aimed enlarging sample, then imaged either diffraction-limited or [13.Chen al.Optical imaging. Expansion microscopy.Science. 347: 543-548Crossref (858) With exception microcopy, specimens, opening avenue understanding dynamics native conditions (Box 1). Overall, four potential uncover as-yet unexplored detail. At same time, their capability requires protocols introduction artifacts tools linkage errors.Box 1Nanometer-scale cells: there yet?FN visualizes entire structures over time 'whole', allowing changes, recommended endoplasmic reticulum, chemical fixation introduces visible [19.Hoffman D.P. al.Correlative three-dimensional block-face whole vitreously frozen cells.Science. 367eaaz5357Crossref (33) our opinion, least connected implementation detailed herein.PhototoxicityPhototoxicity arises absorbed light generates free radicals reactive oxygen species, ultimately causing genomic damage, stress, degradation Figure 1D text). Light both endogenous additionally increase local temperature sample. To some effects, combinations image adaptive illumination [75.Heine al.Adaptive-illumination STED nanoscopy.Proc. Natl. Acad. U. S. A. 114: 9797-9802Crossref (0) Scholar], event-triggered emerging [76.Alvelid al.Event-triggered imaging.Nat. 1268-1275Crossref (15) Furthermore, novel fluorophore classes self-blinking dyes red photoswitchable exist, do require blue excited lower doses [77.Pennacchietti al.Fast reversibly photoswitching live-cell RESOLFT nanoscopy.Nat. 2018; 15: 601-604Crossref (58) Scholar].Low acquisition frequencyLow frequency resolution, but compromises interpretation fast processes. SMLM, limiting factor sufficient number while, scanning-based steps brightness size view. Reducing view spent pixel (dwell time) speed up imaging, decreased contextual SNR. will strongly benefit parallelization deep learning temporal performances.Availability impact cellsThe availability cannot ignored. Some probes drugs bind target affinity, interfering physiology targeted molecule (e.g., phalloidin). An alternative genetically encoded tags or, better, combined genome-editing [78.Bottanelli al.A physiological role ARF1 formation bidirectional tubules Golgi.Mol. 28: 1676-1687Crossref Table 1 text).Imaging depth large viewImaging many samples monolayers. Although feasibility specialized demonstrated [79.Kim al.Oblique-plane tissues small intact animals.Nat. 2019; 16: 853-857Crossref (54) deeper than 10–50 μm methods challenging. engineered illumination, optics, restoration algorithms, multiphoton excitation.Ultimately, best technology selected setup precise question being addressed, controls phototoxic cause damage considered. should done settings less perturbation troubleshooting conditions. For far-red commonly used compared 405-laser irradiance. herein. Phototoxicity Low performances. Availability Imaging excitation. Ultimately, short 'no', all problems solved. Careful consideration given whether necessary questions. determine interest (POI) localized lysosomes mitochondria, POI outer membrane inner matrix, apart. general, strength increased precision biomolecules 1B,C). A simple rule thumb decide needed understand spatial order tens nanometers allows formulation fundamentally hypotheses process under investigation. serendipitous observations made and, cases, revealed new observable [14.Xu K. al.Actin, spectrin, associated form periodic cytoskeletal axons.Science. 2013; 339: 452-456Crossref (888) reason, exploratory Very high-resolution informative, could ignored become challenges Live ultimate goal studies, still due phototoxicity early stage foreseeable relevant near future. fixed already changing field; therefore, concentrate here specimens 1D–F). workflow 'non-live' first optimized avoid artifacts. After antibodies, reveal identities position reporter proximity. relative absolute biologically numbers images (quantitative FN), further measures considerations taken. Box 2, review issues explain why density, stoichiometry, error context. Finally, 3, conditions.Box 2Quantitative FN: versus observed moleculesQuantitative detected matches (or, more precisely, correlates) real Therefore, prerequisites controlled efficiency ability detect fluorophores.A monovalent affinity carrying (or of) reporting 2A Moreover, utilize covalently linked ensure stably labeled preferred. multivalent polyclonal reagents stochastically secondary antibodies approximately one six fluorophores) avoided since correlation between reporters inconsistent.Even ideal case decorated label, possibility exists only decorating label functional. Indeed, inactivated, damaged, during procedure 1E Detecting densely challenging, problem known crowding. When distances single-digit nanometer scale, photophysical interactions occur, resulting undesired alterations properties. happens structure, Förster resonance energy transfer (FRET) H-dimer if separated molecular-scale [80.Ogawa al.H-Type dimer fluorophores: mechanism activatable, vivo imaging.ACS Chem. 2009; 4: 535-546Crossref (153) Notably, reported antibody [81.Helmerich D.A. al.Multiple-labeled behave like emitters buffer.ACS Nano. 14: 12629-12641Crossref (13) serving 'super emitter' while others remain dark state. crowding reversed, physically creating distance Other DNA-PAINT, deal differently modulating concentration probe light-controllable [82.Raymo F.M. Photoactivatable synthetic nanoscale.J. Phys. 2012; 2379-2385Crossref (60) 2D text).While quantification easily achieved, molecule-counting proposed comprehensive recently published challenge [83.Hugelier al.Quantitative microscopy.Annu. Rev. Biophys. 52: 139-160Crossref (1) Importantly, need calibration benchmarked against markers biochemically western blot, liquid chromatography, mass spectrometry).Box 3Fluorophore no one-size-fits-all solutionsA variety developed fulfill requirement technique (Table I). Factors selecting charge, quantum yield, photostability 2C photostable fluorophores, MINFLUX rely switch non-emitting emitting states. mechanisms, refer reader text.The scaffolds cyanines rhodamines. Among cyanines, Alexa Fluor 647 gold standard blinks presence reducing agents UV [84.Berlier J.E. comparison long-wavelength Cy Dyes: bioconjugates.J. Histochem. Cytochem. 2003; 51: 1699-1712Crossref (229) Rhodamines relatively modified tune spectral properties [85.Grimm J.B. general method optimize functionalize red-shifted rhodamine dyes.Nat. 17: 815-821Crossref (85) permeability [86.Lukinavičius near-infrared proteins.Nat. 5: 132-139Crossref (648) equilibrium open closed non-fluorescent forms. regulation latter induces cell-compatible spontaneous blinking Scholar].Other frequently coumarin, oxazine, BODIPY 2E Coumarins among smallest generate variants Stokes shift, advantageous low background [87.Nizamov al.Phosphorylated 3-heteroarylcoumarins nanoscopy.Chem. Eur. 18: 16339-16348Crossref (45) Oxazines [88.Wombacher al.Live-cell trimethoprim conjugates.Nat. 7: 717-719Crossref (282) absorbance, extinction coefficient, 'blink' buffers containing oxidizing agents. valued sharp absorption spectra very yield coefficient [89.Kowada al.BODIPY-based cells.Chem. Soc. 44: 4953-4972Crossref highly hydrophobic nature poor off-switching properties, light-dose photoactivatable make attractive [90.Wijesooriya C.S. localization-based imaging.Angew. Int. Ed. 57 (12685–1268)Crossref (74) Scholar].In after identified, always considering specifications available instrument lasers detectors). choice also driven needs, multicolor autofluorescence reduce background, counting naked may themselves affinities lipophilic stain membranes). evaluated designing experiments, (reporters) without targeting moiety whenever 2B recommend inexperienced users consult expert select dye application.Table IProperties dyesFluorophore classCoumarinsRhodaminesCyaninesBODIPYsOxazinesCommercial examplesAlexa 350, Pacific BlueAlexa 488, silicon-rhodamine, TMRAlexa 647, 555, Cy5BODIPY FL, TMRAtto 655, Atto 680Spectral range (nm)360–700500–750500–1000500–700600–750Extinction (cm–1M–1)15 000–60 00080 000–150 000130 000–250 00060 000–100 000110 000–130 000Quantum yield0.4–0.90.1–0.90.1–0.60.8–0.90.1–0.6Photostability+++++++++++Compatibility methodsSTED, SMLMSTED, MINFLUX, microscopySTED, SMLMSMLM Open table tab Quantitative fluorophores. inconsistent. Even sin

Language: Английский

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Fluorescent probes for bioimaging of potential biomarkers in Parkinson's disease

Chemical Society Reviews, Journal Year: 2020, Volume and Issue: 50(2), P. 1219 - 1250

Published: Dec. 8, 2020

Parkinson's disease (PD), as the second most common neurodegenerative disease, is caused by complex pathological processes and currently remains very difficult to treat. PD brings great distress patients imposes a heavy economic burden on society. The number of growing aging population increases worldwide. Therefore, it crucial develop new tools for aiding early diagnosis treatment PD. significant features involved in include abnormal accumulation α-synuclein, metal ion dyshomeostasis, oxidative stress, mitochondrial dysfunction neurotransmitter deficiencies. In recent years, fluorescent probes have emerged powerful bioimaging tool with potential help understand via detection monitoring features. this review, we comprehensively summarize design working mechanisms along their applications various biomarkers. We also discuss current limitations provide perspectives how these can be overcome better suitable application clinical trials future. hope that review provides valuable information guidance development used clinically contributes efficient drugs

Language: Английский

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Development of nanobody-horseradish peroxidase-based sandwich ELISA to detect Salmonella Enteritidis in milk and in vivo colonization in chicken

Journal of Nanobiotechnology, Journal Year: 2022, Volume and Issue: 20(1)

Published: March 31, 2022

Salmonella Enteritidis (S. Enteritidis) being one of the most prevalent foodborne pathogens worldwide poses a serious threat to public safety. Prevention zoonotic infectious disease and controlling risk transmission S. Enteriditidis critically requires evolution rapid sensitive detection methods. The methods based on nucleic acid conventional antibodies are fraught with limitations. Many these limitations can be circumvented using natural nanobodies which endowed characteristics, such as high affinity, thermal stability, easy production, especially higher diversity. This study aimed select special against for developing an improved nanobody-horseradish peroxidase-based sandwich ELISA detect in practical sample. peroxidase fusions help eliminating use secondary labeled horseradish peroxidase, reduce time experiment. Moreover, novel developed this used specifically rapidly sensitivity.This screened four from immunized nanobody library, after rounds screening, phage display technology. Subsequently, were successfully expressed prokaryotic eukaryotic expression systems, respectively. A employing SE-Nb9 peroxidase-Nb1 pair capture Enteritidis, respectively, was found possess limit 5 × 104 colony forming units (CFU)/mL. In established immunoassay, 8 h-enrichment enabled up approximately 10 CFU/mL milk samples. Furthermore, we investigated colonization distribution infected chicken assay, showing that could subsist almost all parts intestinal tract. These results agreement obtained real-time PCR plate culture. liver identified colonized quite several Enteriditidis, indicating infection outside tract.This newly antibody spike sample analyze chicken. demonstrated assay is applicable detecting spiked rapid, specific, way. Meanwhile, challenged indicate it promising tool monitoring poultry products. Importantly, SE-Nb1-vHRP directly bind captured by SE-Nb9, reducing commercial shortening time. short, ushers great prospects food safety control further production.

Language: Английский

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Research Progress and Applications of Multivalent, Multispecific and Modified Nanobodies for Disease Treatment

Frontiers in Immunology, Journal Year: 2022, Volume and Issue: 12

Published: Jan. 18, 2022

Recombinant antibodies such as nanobodies are progressively demonstrating to be a valid alternative conventional monoclonal also for clinical applications. Furthermore, they do not solely represent substitute but their unique features allow expanding the applications of biotherapeutics and changes pattern disease treatment. Nanobodies possess double advantage being small simple engineer. This combination has promoted extremely diversified approaches design nanobody-based constructs suitable particular Both format geometry possibilities functionalization strategies have been widely explored provide macromolecules with better efficacy respect single or combination. Nanobody multimers nanobody-derived reagents were developed image contrast several cancer diseases shown effectiveness in animal models. Their capacity block more independent signaling pathways simultaneously is considered critical avoid tumor resistance, whereas mass these multimeric compounds still remains significantly smaller than that an IgG, enabling deeper penetration solid tumors. When applied CAR-T cell therapy, can effectively improve specificity by targeting multiple epitopes consequently reduce side effects. represents great potential treating malignant lymphomas, acute myeloid leukemia, lymphoblastic myeloma Apart from treatment, multispecific drugs imaging built nanobody blocks demonstrated value detecting tackling neurodegenerative, autoimmune, metabolic, infectious antidotes toxins. In particular, multi-paratopic recently passive immunization against SARS-CoV-2 goal impairing variant survival due resistance epitopes. Given enormous research activity field, it expected molecules will undergo late trials next future. Systematic Review Registration.

Language: Английский

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Visualizing proteins by expansion microscopy

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2022, Volume and Issue: unknown

Published: Aug. 5, 2022

Abstract Fluorescence imaging is one of the most versatile and widely-used tools in biology 1 . Although techniques to overcome diffraction barrier were introduced more than two decades ago, nominal attainable resolution kept improving 2, 3 , fluorescence microscopy still fails image morphology single proteins or small molecular complexes, either purified a cellular context 4, 5 Here we report solution this problem, form o ne-step n anoscale e xpansion (ONE) microscopy. We combined 10-fold axial expansion specimen (1000-fold by volume) with fluctuation analysis 6, 7 enable description cultured cells, tissues, viral particles, complexes proteins. At level, using immunostaining, our technology revealed detailed nanoscale arrangements synaptic proteins, including quasi-regular organisation PSD95 clusters. molecule upon main chain fluorescent labelling, could visualise shape individual membrane soluble Moreover, conformational changes undergone ∼17 kDa protein calmodulin Ca 2+ binding readily observable. also imaged classified aggregates cerebrospinal fluid samples from Parkinson’s Disease (PD) patients, which represents promising new development towards improved PD diagnosis. ONE compatible conventional microscopes can be performed software provide here as free, open-source package. This bridges gap between high-resolution structural light microscopy, provides avenue for discoveries medicine.

Language: Английский

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One-step nanoscale expansion microscopy reveals individual protein shapes

Nature Biotechnology, Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 9, 2024

The attainable resolution of fluorescence microscopy has reached the subnanometer range, but this technique still fails to image morphology single proteins or small molecular complexes. Here, we expand specimens at least tenfold, label them with conventional fluorophores and light microscopes, acquiring videos in which analyze fluctuations. One-step nanoscale expansion (ONE) enables visualization shapes individual membrane soluble proteins, achieving around 1-nm resolution. We show that conformational changes are readily observable, such as those undergone by ~17-kDa protein calmodulin upon Ca2+ binding. ONE is also applied clinical samples, analyzing aggregates cerebrospinal fluid from persons Parkinson disease, potentially aiding disease diagnosis. This technology bridges gap between high-resolution structural biology techniques microscopy, providing new avenues for discoveries medicine. Combining super-resolution radial fluctuations captures proteins.

Language: Английский

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Revolutionizing Cancer Treatment: Recent Advances in Immunotherapy

Biomedicines, Journal Year: 2024, Volume and Issue: 12(9), P. 2158 - 2158

Published: Sept. 23, 2024

Cancer immunotherapy has emerged as a transformative approach in oncology, utilizing the body’s immune system to specifically target and destroy malignant cells. This review explores scope impact of various immunotherapeutic strategies, including monoclonal antibodies, chimeric antigen receptor (CAR)-T cell therapy, checkpoint inhibitors, cytokine therapeutic vaccines. Monoclonal such Rituximab Trastuzumab, have revolutionized treatment paradigms for lymphoma breast cancer by offering targeted interventions that reduce off-target effects. CAR-T therapy presents potentially curative option refractory hematologic malignancies, although challenges remain effectively treating solid tumors. Checkpoint inhibitors redefined management cancers like melanoma lung cancer; however, managing immune-related adverse events ensuring durable responses are critical areas focus. Cytokine continues play vital role modulating response, with advancements engineering improving specificity reducing systemic toxicity. Therapeutic vaccines, particularly mRNA-based represent frontier personalized treatment, aiming generate robust, long-lasting against tumor-specific antigens. Despite these advancements, field faces significant challenges, resistance, tumor heterogeneity, immunosuppressive microenvironment. Future research should address obstacles through emerging technologies, next-generation Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-based gene editing, AI-driven drug discovery. By integrating novel approaches, holds promise more durable, less toxic, highly options, ultimately patient outcomes survival rates.

Language: Английский

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Unveiling the Cutting Edge: Recent Developments in α-Synuclein Nano-biosensor Technology for Precision Diagnosis of Parkinson’s Disease

BioNanoScience, Journal Year: 2025, Volume and Issue: 15(1)

Published: Jan. 7, 2025

Language: Английский

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