Picomolar Biosensing and Conformational Analysis Using Artificial Bidomain Proteins and Terbium-to-Quantum Dot Förster Resonance Energy Transfer

Léger, Corentin; Yahia-Ammar, Akram; Susumu, Kimihiro; Medintz, Igor L.; Urvoas, Agathe; Valerio-Lepiniec, Marie; Minard, Philippe; Hildebrandt, Niko

doi:10.1021/acsnano.0c01410

Cited by 26 publications

(25 citation statements)

References 59 publications

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“…The ubiquity of antibodies in cell imaging arises from the ability to select specific, high-affinity probes against a wide range of receptors and other cellular components. , Advances in Ab selection methods and engineering have both expanded the range of possible targets and enabled the development of miniaturized Ab designs with specific conjugation handles that avoid some of the pitfalls of traditional Ab labeling techniques, such as breakdown under physiological conditions, either for noncovalent − , or equilibrium covalent ,, interactions. Common labeling techniques that require chemical modification of the Ab, such as oxidation or reduction, also run the risk of diminishing Ab–ligand affinity or Ab stability and need to be evaluated on a case-by-case basis. − , For synthesis of NP-Ab complexes, most reactions adapted from organic fluorophore chemistry, such as lysine side chain modification, are far more difficult to control with the multivalent reactivity of both Ab , and NP surfaces, , which presents the risk of significant aggregation.…”

Section: Resultsmentioning

confidence: 99%

“…[17][18][19][20] For Ab-based imaging applications, inorganic NPs have adapted organic fluorophore bioconjugation reactions, 14,21,22 but difficulties in controlling immunoconjugate size, stoichiometry, Ab orientation, and stability have limited the broad utility of Ab-NP conjugates. [25][26][27][28][29][30][31] Newer covalent reactions that address these issues may be useful in expanding the scope of protein-NP conjugates in bioimaging, 32 although these bimolecular reactions typically require higher reactant concentration than is possible for relatively large proteins and colloidal nanoparticles. One exception is the engineered split protein SpyCatcher/SpyTag, 33,34 in which the components bind to one another with nanomolar affinity before forming stable isopeptide bonds, and which is emerging as a versatile system for the controlled and stable conjugation of proteins to NPs.…”

mentioning

confidence: 99%

“…Inorganic nanoparticles (NPs) possess optical properties that make them particularly well-suited for live cell imaging. Compared to organic or protein-based fluorophores, semiconductor quantum dots (QDs) have larger optical cross sections, increased photostability, and broadband excitation spectra. − Similarly, upconverting nanoparticles (UCNPs), which emit at visible wavelengths after absorption of multiple near-infrared (NIR) photons, show no overlap with cellular autofluorescence, no measurable blinking or photobleaching at single-molecule powers, and can be imaged millimeters into tissue with exceptionally low laser fluences. − For Ab-based imaging applications, inorganic NPs have adapted organic fluorophore bioconjugation reactions, ,, but difficulties in controlling immunoconjugate size, stoichiometry, Ab orientation, and stability have limited the broad utility of Ab-NP conjugates. − Bioorthogonal covalent reactions that address these issues may be useful in expanding the scope of protein–NP conjugates in bioimaging, although these bimolecular reactions typically require higher reactant concentrations than are possible for relatively large proteins and colloidal nanoparticles. An exception is the engineered split protein SpyCatcher/SpyTag, , in which the components bind to one another with nanomolar affinity before forming stable isopeptide bonds and which is emerging as a versatile system for the controlled, stable conjugation of proteins to NPs. , …”

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Immunotargeting of Nanocrystals by SpyCatcher Conjugation of Engineered Antibodies

et al. 2021

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Inorganic nanocrystals such as quantum dots (QDs) and upconverting nanoparticles (UCNPs) are uniquely suited for quantitative live-cell imaging and are typically functionalized with ligands to study specific receptors or cellular targets. Antibodies (Ab) are among the most useful targeting reagents owing to their high affinities and specificities, but common nanocrystal labeling methods may orient Ab incorrectly, be reversible or denaturing, or lead to Ab-NP complexes too large for some applications. Here, we show that SpyCatcher proteins, which bind and spontaneously form covalent isopeptide bonds with cognate SpyTag peptides, can conjugate engineered Ab to nanoparticle surfaces with control over stability, orientation, and stoichiometry. Compact SpyCatcher-functionalized QDs and UCNPs may be labeled with short-chain variable fragment Ab (scFv) engineered to bind urokinase-type plasminogen activator receptors (uPAR) that are overexpressed in many human cancers. Confocal imaging of anti-uPAR scFv-QD conjugates shows the Ab mediates specific binding and internalization by breast cancer cells expressing uPAR. Time-lapse imaging of photostable scFv-UCNP conjugates show that Ab binding causes uPAR internalization with a ∼20-minute half-life on the cell surface, and uPAR is internalized to endolysosomal compartments distinct from general membrane stains and without significant recycling to the cell surface. The controlled and stable conjugation of engineered Ab to NPs enables targeting of diverse receptors for live-cell study of their distribution, trafficking, and physiology.

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Section: Resultsmentioning

confidence: 99%

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Immunotargeting of Nanocrystals by SpyCatcher Conjugation of Engineered Antibodies

et al. 2021

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“…25 In addition, QDs can act as fluorescence resonance energy transfer (FRET) donors and acceptors to construct various biosensors for homogeneous detection of nucleic acids and proteins. 26,27 The integration of single-molecule detection with the QDs endows the biosensors with distinct characteristics of high sensitivity and low sample consumption. 28 The isothermal RCA reaction may generate very long single-stranded DNAs (ssDNAs) with tandem repeats, and it has been used for miRNA and cancer cell detection.…”

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confidence: 99%

“…Moreover, the large surface area of QDs facilitates the covalent conjugation of various biorecognition molecules for the preparation of fluorescent probes . In addition, QDs can act as fluorescence resonance energy transfer (FRET) donors and acceptors to construct various biosensors for homogeneous detection of nucleic acids and proteins. , The integration of single-molecule detection with the QDs endows the biosensors with distinct characteristics of high sensitivity and low sample consumption . The isothermal RCA reaction may generate very long single-stranded DNAs (ssDNAs) with tandem repeats, and it has been used for miRNA and cancer cell detection. , Herein, we developed a single QD-mediated FRET nanosensor with the integration of multiple primer generation rolling circle amplification (MPG-RCA) for sensitive detection of KRAS G12D mutation in cancer cells.…”

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Development of a Single Quantum Dot-Mediated FRET Nanosensor for Sensitive Detection of Single-Nucleotide Polymorphism in Cancer Cells

Liu

et al. 2021

Anal. Chem.

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Single-nucleotide polymorphisms (SNPs) are important hallmarks of human diseases. Herein, we develop a single quantum dot (QD)-mediated fluorescence resonance energy transfer (FRET) nanosensor with the integration of multiple primer generation rolling circle amplification (MPG-RCA) for sensitive detection of SNPs in cancer cells. This assay involves only a linear padlock probe for MPG-RCA. The presence of a mutant target facilitates the circularization of linear padlock probes to initiate RCA, producing three short single-stranded DNAs (ssDNAs) with the assistance of nicking endonuclease. The resulting ssDNAs can function as primers to induce cyclic MPG-RCA, resulting in the exponential amplification and generation of large numbers of linker probes. The linker probes can subsequently hybridize with the Cy5-labeled reporter probes and the biotinylated capture probes to obtain the sandwich hybrids. The assembly of these sandwich hybrids on the 605 nm-emission quantum dot (605QD) generates the 605QD−oligonucleotide−Cy5 nanostructures, resulting in efficient FRET from the 605QD to Cy5. This nanosensor is free from both the complicated probe design and the exogenous primers and has distinct advantages of high amplification efficiency, zero background signal, good specificity, and high sensitivity. It can detect SNPs with a large dynamic range of 8 orders of magnitude and a detection limit of 5.41 × 10 −20 M. Moreover, this nanosensor can accurately distinguish as low as 0.001% mutation level from the mixtures, which cannot be achieved by previously reported methods. Furthermore, it can discriminate cancer cells from normal cells and even quantify SNP at the single-cell level.

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A Nanobody‐on‐Quantum Dot Displacement Assay for Rapid and Sensitive Quantification of the Epidermal Growth Factor Receptor (EGFR)

Doulkeridou

et al. 2022

Angewandte Chemie

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Biosensing approaches that combine small, engineered antibodies (nanobodies) with nanoparticles are often complicated. Here, we show that nanobodies with different C‐terminal tags can be efficiently attached to a range of the most widely used biocompatible semiconductor quantum dots (QDs). Direct implementation into simplified assay formats was demonstrated by designing a rapid and wash‐free mix‐and‐measure immunoassay for the epidermal growth factor receptor (EGFR). Terbium complex (Tb)‐labeled hexahistidine‐tagged nanobodies were specifically displaced from QD surfaces via EGFR‐nanobody binding, leading to an EGFR concentration‐dependent decrease of the Tb‐to‐QD Förster resonance energy transfer (FRET) signal. The detection limit of 80±20 pM (16±4 ng mL−1) was 3‐fold lower than the clinical cut‐off concentration for soluble EGFR and up to 10‐fold lower compared to conventional sandwich FRET assays that required a pair of different nanobodies.

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Picomolar Biosensing and Conformational Analysis Using Artificial Bidomain Proteins and Terbium-to-Quantum Dot Förster Resonance Energy Transfer

Cited by 26 publications

References 59 publications

Immunotargeting of Nanocrystals by SpyCatcher Conjugation of Engineered Antibodies

Immunotargeting of Nanocrystals by SpyCatcher Conjugation of Engineered Antibodies

Development of a Single Quantum Dot-Mediated FRET Nanosensor for Sensitive Detection of Single-Nucleotide Polymorphism in Cancer Cells

A Nanobody‐on‐Quantum Dot Displacement Assay for Rapid and Sensitive Quantification of the Epidermal Growth Factor Receptor (EGFR)

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