Determination of the Labeling Density of Fluorophore–Biomolecule Conjugates with Absorption Spectroscopy

Grabolle, Markus; Brehm, Robert; Pauli, Jutta; Dees, Franziska M; Hilger, Ingrid; Resch‐Genger, Ute

doi:10.1021/bc2003428

Cited by 30 publications

(27 citation statements)

References 41 publications

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“…Each monomer was characterized by absorption spectroscopy studies in ethanol (Table S1 and Figure S1 of the SI). Both the absorption coefficient at λ max and the integral absorption coefficient 39 were determined for each dye label and found to be close to the values measured for a commercially available Nile Blue dye precursor (95% purity, laser grade), which suggests high purities for these Nile Blue-based monomers (Table S1 of the SI). …”

Section: Resultssupporting

confidence: 68%

Nile Blue-Based Nanosized pH Sensors for Simultaneous Far-Red and Near-Infrared Live Bioimaging

et al. 2013

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Diblock copolymer vesicles are tagged with pH-responsive Nile Blue-based labels and used as a new type of pH-responsive colorimetric/fluorescent biosensor for far-red and near-infrared imaging of live cells. The diblock copolymer vesicles described herein are based on poly(2-(methacryloyloxy)ethyl phosphorylcholine-block-2-(diisopropylamino)ethyl methacrylate) [PMPC-PDPA]: the biomimetic PMPC block is known to facilitate rapid cell uptake for a wide range of cell lines, while the PDPA block constitutes the pH-responsive component that enables facile vesicle self-assembly in aqueous solution. These biocompatible vesicles can be utilized to detect interstitial hypoxic/acidic regions in a tumor model via a pH-dependent colorimetric shift. In addition, they are also useful for selective intracellular staining of lysosomes and early endosomes via subtle changes in fluorescence emission. Such nanoparticles combine efficient cellular uptake with a pH-responsive Nile Blue dye label to produce a highly versatile dual capability probe. This is in marked contrast to small molecule dyes, which are usually poorly uptaken by cells, frequently exhibit cytotoxicity, and are characterized by intracellular distributions invariably dictated by their hydrophilic/hydrophobic balance.

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Section: Resultssupporting

confidence: 68%

Nile Blue-Based Nanosized pH Sensors for Simultaneous Far-Red and Near-Infrared Live Bioimaging

et al. 2013

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“…Values are gathered in Ta ble 2 and comparedw ith those determined by the standard spectrophotometric method. [22] Satisfactory F/P valuesw ithin the range 1.4-3.6 were obtained, depending on the excess of "Borico" dye and the pH of the labeling mixture used, thus substantiating the covalent fluorescentl abeling. Variances in the results (mainly for fluorescent conjugates BSA-8 prepared at pH 7.0) obtained using the two methods can be primarily explained as follows:1 )for the photometric method, molar absorptivity determined for the free "7-hydroxy-Borico" dye 8 was used for the quantification, but its covalentb inding to the protein changed the value of this spectral parameter,a nd 2) the presence of residual amounts of free "7-hydroxy-Borico" dye 8 not removedb yu ltracentrifugation and possibly adsorbed on the protein.…”

Section: Fluorogenic Reactivity Of "7-hydroxy-borico" Derivativesmentioning

confidence: 68%

New 3‐(Heteroaryl)‐2‐iminocoumarin‐based Borate Complexes: Synthesis, Photophysical Properties, and Rational Functionalization for Biosensing/Biolabeling Applications

Roubinet

Massif

Moreau

et al. 2015

Chemistry A European J

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Members of a series of boron difluoride complexes with 3-(heteroaryl)-2-iminocoumarin ligands bearing both a phenolic hydroxyl group (acting as a fluorogenic center) and an N-aryl substituent (acting as a stabilizing moiety) have been synthesized in good yields by applying a straightforward two-step method. These novel fluorogenic dyes belong to the family of "Boricos" (D. Frath et al., Chem. Commun.- 2013, 49, 4908-4910) and are the first examples of phenol-based fluorophores of which the photophysical properties in the green-yellow spectral range are dramatically improved by N,N-chelation of a boron atom. Modulation of their fluorescence properties through reversible chemical modification of their phenol moieties has been demonstrated by the preparation of the corresponding 2,4-dinitrophenyl (DNP) ethers, which led to a dramatic "OFF-ON" fluorescence response upon reaction with thiols. Additionally, to expand the scope of these "7-hydroxy-Borico" derivatives, particularly in biolabeling, amine or carboxylic acid functionalities amenable to (bio)conjugation have been introduced within their scaffold. Their utility has been demonstrated in the preparation of fluorescent bovine serum albumin (BSA) conjugates and "Borico"-DOTA-like scaffolds in an effort to design novel monomolecular multimodal fluorescence- radioisotope imaging agents.

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“… 1 Fluorescent molecules can be used as molecular environmental probes by selecting dyes with strong responses to, for example, pH, 2 viscosity, 3 polarizability, 4 elasticity, 5 and polarity; 6 however, in applications where the fluorescent intensity is to serve as a proxy for the abundance of the labeled molecule, environmental sensitivity is a liability that can result in reduced measurement accuracy. 7 The cyanine dyes Cy3 and Cy5 are among the most widely used and versatile 8 oligonucleotide labels in, e.g., microarray experiments, fluorescent in situ hybridization (FISH), real-time PCR (RT-PCR), and FRET studies 9 , 10 and are considered to be relatively environmentally insensitive. 11 However, Cy3 and Cy5 consist of two indole rings connected by three or five carbon polymethine bridges which can undergo cis – trans isomerization from the first excited singlet state which competes with fluorescence.…”

Section: Introductionmentioning

confidence: 99%

Sequence-Dependent Fluorescence of Cy3- and Cy5-Labeled Double-Stranded DNA

2016

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The fluorescent intensity of Cy3 and Cy5 dyes is strongly dependent on the nucleobase sequence of the labeled oligonucleotides. Sequence-dependent fluorescence may significantly influence the data obtained from many common experimental methods based on fluorescence detection of nucleic acids, such as sequencing, PCR, FRET, and FISH. To quantify sequence dependent fluorescence, we have measured the fluorescence intensity of Cy3 and Cy5 bound to the 5′ end of all 1024 possible double-stranded DNA 5mers. The fluorescence intensity was also determined for these dyes bound to the 5′ end of fixed-sequence double-stranded DNA with a variable sequence 3′ overhang adjacent to the dye. The labeled DNA oligonucleotides were made using light-directed, in situ microarray synthesis. The results indicate that the fluorescence intensity of both dyes is sensitive to all five bases or base pairs, that the sequence dependence is stronger for double- (vs single-) stranded DNA, and that the dyes are sensitive to both the adjacent dsDNA sequence and the 3′-ssDNA overhang. Purine-rich sequences result in higher fluorescence. The results can be used to estimate measurement error in experiments with fluorescent-labeled DNA, as well as to optimize the fluorescent signal by considering the nucleobase environment of the labeling cyanine dye.

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Determination of the Labeling Density of Fluorophore–Biomolecule Conjugates with Absorption Spectroscopy

Cited by 30 publications

References 41 publications

Nile Blue-Based Nanosized pH Sensors for Simultaneous Far-Red and Near-Infrared Live Bioimaging

Nile Blue-Based Nanosized pH Sensors for Simultaneous Far-Red and Near-Infrared Live Bioimaging

New 3‐(Heteroaryl)‐2‐iminocoumarin‐based Borate Complexes: Synthesis, Photophysical Properties, and Rational Functionalization for Biosensing/Biolabeling Applications

Sequence-Dependent Fluorescence of Cy3- and Cy5-Labeled Double-Stranded DNA

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