Dark Quencher Research Articles

Hemin as a General Static Dark Quencher for Constructing Heme Oxygenase‐1 Fluorescent Probes

AbstractThe development of small‐molecule probes suitable for live‐cell applications remains challenging yet highly desirable. We report the first fluorescent probe, RBH, for imaging the heme oxygenase‐1 (HO‐1) activity in live cells after discovering hemin as a universal dark quencher. Hemin works via a static quenching mechanism and shows high quenching efficiency (>97 %) with fluorophores across a broad spectrum (λex=400–700 nm). The favorable properties of RBH (e.g. long excitation/emission wavelengths, fast response rate and high magnitude of signal increase) enable its use for determining HO‐1 activity in complex biological samples. As HO‐1 is involved in regulating antioxidant defence, iron homeostasis and gasotransmitter carbon monoxide production, we expect RBH to be a powerful tool for dissecting its functions. Also, the discovery of hemin as a general static dark quencher provides a straightforward strategy for constructing novel fluorescent probes for diverse biological species.

Hemin as a General Static Dark Quencher for Constructing Heme Oxygenase-1 Fluorescent Probes.

The development of small-molecule probes suitable for live-cell applications remains challenging yet highly desirable. We report the first fluorescent probe, RBH, for imaging the heme oxygenase-1 (HO-1) activity in live cells after discovering hemin as a universal dark quencher. Hemin works via a static quenching mechanism and shows high quenching efficiency (>97 %) with fluorophores across a broad spectrum (λex =400-700 nm). The favorable properties of RBH (e.g. long excitation/emission wavelengths, fast response rate and high magnitude of signal increase) enable its use for determining HO-1 activity in complex biological samples. As HO-1 is involved in regulating antioxidant defence, iron homeostasis and gasotransmitter carbon monoxide production, we expect RBH to be a powerful tool for dissecting its functions. Also, the discovery of hemin as a general static dark quencher provides a straightforward strategy for constructing novel fluorescent probes for diverse biological species.

Shrinking gate fluorescence correlation spectroscopy yields equilibrium constants and separates photophysics from structural dynamics

Fluorescence correlation spectroscopy is a versatile tool for studying fast conformational changes of biomolecules especially when combined with Förster resonance energy transfer (FRET). Despite the many methods available for identifying structural dynamics in FRET experiments, the determination of the forward and backward transition rate constants and thereby also the equilibrium constant is difficult when two intensity levels are involved. Here, we combine intensity correlation analysis with fluorescence lifetime information by including only a subset of photons in the autocorrelation analysis based on their arrival time with respect to the excitation pulse (microtime). By fitting the correlation amplitude as a function of microtime gate, the transition rate constants from two fluorescence-intensity level systems and the corresponding equilibrium constants are obtained. This shrinking-gate fluorescence correlation spectroscopy (sg-FCS) approach is demonstrated using simulations and with a DNA origami-based model system in experiments on immobilized and freely diffusing molecules. We further show that sg-FCS can distinguish photophysics from dynamic intensity changes even if a dark quencher, in this case graphene, is involved. Finally, we unravel the mechanism of a FRET-based membrane charge sensor indicating the broad potential of the method. With sg-FCS, we present an algorithm that does not require prior knowledge and is therefore easily implemented when an autocorrelation analysis is carried out on time-correlated single-photon data.

Bringing to light the physiological and pathological firing patterns of human induced pluripotent stem cell-derived neurons using optical recordings.

Human induced pluripotent stem cells (hiPSCs) are a promising approach to study neurological and neuropsychiatric diseases. Most methods to record the activity of these cells have major drawbacks as they are invasive or they do not allow single cell resolution. Genetically encoded voltage indicators (GEVIs) open the path to high throughput visualization of undisturbed neuronal activity. However, conventional GEVIs perturb membrane integrity through inserting multiple copies of transmembrane domains into the plasma membrane. To circumvent large add-ons to the plasma membrane, we used a minimally invasive novel hybrid dark quencher GEVI to record the physiological and pathological firing patterns of hiPSCs-derived sensory neurons from patients with inherited erythromelalgia, a chronic pain condition associated with recurrent attacks of redness and swelling in the distal extremities. We observed considerable differences in action potential firing patterns between patient and control neurons that were previously overlooked with other recording methods. Our system also performed well in hiPSC-derived forebrain neurons where it detected spontaneous synchronous bursting behavior, thus opening the path to future applications in other cell types and disease models including Parkinson's disease, Alzheimer's disease, epilepsy, and schizophrenia, conditions associated with disturbances of neuronal activity and synchrony.

Small-Molecule Quenchers for Förster Resonance Energy Transfer: Structure, Mechanism, and Applications.

For fluorogenic probes, Förster resonance energy transfer (FRET) is one of the most widely used mechanisms to realize the detection of biochemical activities. Dark quenchers relax from the excited state to the ground state non-radiatively, and are promising alternatives to fluorescent FRET acceptors. Small-molecule (dark) quenchers have been widely used as acceptors in FRET-based probes to monitor various physiological processes with minimum background signal. Herein, we summarize the relevant advances of small-molecule quenchers that are used in FRET-based probes. This Review is intended to provide suggestions regarding the rationale design and selection of appropriate fluorophore-quencher FRET pairs, which are fully compatible with challenging analytical applications in various biological systems. Finally, an outlook of the future biomedical applications and developments of this field is presented.

Small‐Molecule Quenchers for Förster Resonance Energy Transfer: Structure, Mechanism, and Applications

AbstractFor fluorogenic probes, Förster resonance energy transfer (FRET) is one of the most widely used mechanisms to realize the detection of biochemical activities. Dark quenchers relax from the excited state to the ground state non‐radiatively, and are promising alternatives to fluorescent FRET acceptors. Small‐molecule (dark) quenchers have been widely used as acceptors in FRET‐based probes to monitor various physiological processes with minimum background signal. Herein, we summarize the relevant advances of small‐molecule quenchers that are used in FRET‐based probes. This Review is intended to provide suggestions regarding the rationale design and selection of appropriate fluorophore–quencher FRET pairs, which are fully compatible with challenging analytical applications in various biological systems. Finally, an outlook of the future biomedical applications and developments of this field is presented.

Synthesis and characterization of N-rich fluorescent bio-dots as a reporter in the design of dual-labeled FRET probe for TaqMan PCR: A feasibility study.

DNA-based analytical techniques have provided an advantageous sensing assay in the realm of biotechnology. Bio-inspired fluorescent nanodots are a novel type of biological staining agents with excellent optical properties widely used for cellular imaging and diagnostics. In the present research, we successfully synthesized bio-dots with excellent optical properties and high-quantum yield from DNA sodium salt through the hydrothermal method. We conjugated the bio-dots with 3' Eclipse Dark Quencher (Eclipse)-labeled single-strand oligodeoxyribonucleotide according to carbodiimide chemistry, to design a fluorescence resonance energy transfer (FRET) probe. The results confirmed the prosperous synthesis and surface functionalization of the bio-dot. Analysis of size, zeta potential, and FTIR spectroscopy verified successful bioconjugation of the bio-dots with probes. UV-visibility analysis and fluorescence intensity profile of the bio-dot and bio-dot@probes represented a concentration-dependent quenching of fluorescent signal of bio-dot by Eclipse after probe conjugation. The results demonstrated that TaqMan PCR was not feasible using the designed bio-dot@probes. Our results indicated that bio-dot can be used as an efficient fluorescent tag in the design of fluorescently labeled oligonucleotides with high biocompatibility and optical features.

A sensitive and facile microRNA detection based on CRISPR-Cas12a coupled with strand displacement amplification

MicroRNAs (miRNAs) are important biomarkers that are closely associated with certain diseases. The detection of miRNA is critical because it provides the necessary information for Disease Diagnosis. In this study, we achieved miRNA determination by coupling the CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated) system with strand displacement amplification (SDA). In the experiment, miRNA was used as the initiator of SDA, and the activator of Cas12a nuclease activity was amplified by SDA. Subsequently, the unique nuclease activity of Cas12a was exploited to carry out trans cleaving on the ssDNA reporting probe modified with carboxyfluorescein(FAM) and BHQ1(dark Quencher: 480–580 nm) to achieve a signal output. In addition to chain design and reaction simplification, this method is lofty sensitive and selective for the determination of miRNA with a good linear range of 250 fmol·L−1 ∼ 40 pmol·L−1, the detection limit of 150 fmol·L−1 (S/N = 3), and the method showed good recovery in spiked human serum. Overall, this method is expected to be applied to diagnosis with miRNA biomarkers because of its rapidity, high sensitivity, and high selectivity.

Enzymatic Beacons for Specific Sensing of Dilute Nucleic Acid.

Enzymatic beacons, or E-beacons, are 1 : 1 bioconjugates of the nanoluciferase enzyme linked covalently at its C-terminus to hairpin forming ssDNA equipped with a dark quencher. We prepared E-beacons biocatalytically using HhC, the promiscuous Hedgehog C-terminal protein-cholesterol ligase. HhC attached nanoluciferase site-specifically to mono-sterylated hairpin oligonucleotides, called steramers. Three E-beacon dark quenchers were evaluated: Iowa Black, Onyx-A, and dabcyl. Each quencher enabled sensitive, sequence-specific nucleic acid detection through enhanced E-beacon bioluminescence upon target hybridization. We assembled prototype dabcyl-quenched E-beacons specific for SARS-CoV-2. Targeting the E484 codon of the virus Spike protein, E-beacons (80×10-12 M) reported wild-type SARS-CoV-2 nucleic acid at ≥1×10-9 M by increased bioluminescence of 8-fold. E-beacon prepared for the SARS-CoV-2 E484K variant functioned with similar sensitivity. Both E-beacons could discriminate their target from the E484Q mutation of the SARS-CoV-2 Kappa variant. Along with mismatch specificity, E-beacons are two to three orders of magnitude more sensitive than synthetic molecular beacons.

Chemoselective Attachment of the Water-Soluble Dark Quencher Hydrodabcyl to Amino Groups in Peptides and Preservation of Its Spectroscopic Properties over a Wide pH Range.

The water-soluble quencher hydrodabcyl can be activated as an N-succinimidyl ester that is readily accessible from crude hydrodabcyl and storable for a long time. With primary and secondary amines, it reacts swiftly and chemoselectively, even in the presence of other competing nucleophiles such as those typically present in natural peptides. One of the three phenolic OH groups of hydrodabcyl is amenable to selective mono-Boc protection resulting in reduced polarity, advantageous to its further use in organic synthesis. The advantages of hydrodabcyl over dabcyl in spectrometric applications are exemplified by the pH dependence of its absorbance spectra.

Monitoring Human Neutrophil Activation by a Proteinase 3 Near-Infrared Fluorescence Substrate-Based Probe.

A near-infrared fluorescent (NIRF) substrate-based probe (SBP) was conceived to monitor secreted human proteinase 3 (hPR3) activity. This probe, called pro3-SBP, is shaped by a fused peptide hairpin loop structure, which associates a hPR3 recognition domain (Val-Ala-Asp-Nva-Ala-Asp-Tyr-Gln, where Nva is norvaline) and an electrostatic zipper (consisting of complementary polyanionic (d-Glu)5 and polycationic (d-Arg)5 sequences) in close vicinity of the N- and C-terminal FRET couple (fluorescent donor, sulfoCy5.5; dark quencher, QSY21). Besides its subsequent stability, no intermolecular fluorescence quenching was detected following its complete hydrolysis by hPR3, advocating that pro3-SBP could further afford unbiased imaging. Pro3-SBP was specifically hydrolyzed by hPR3 (kcat/Km= 440 000 ± 5500 M-1·s-1) and displayed a sensitive detection threshold for hPR3 (subnanomolar concentration range), while neutrophil elastase showed a weaker potency. Conversely, pro3-SBP was not cleaved by cathepsin G. Pro3-SBP was successfully hydrolyzed by conditioned media of activated human neutrophils but not by quiescent neutrophils. Moreover, unlike unstimulated neutrophils, a strong NIRF signal was specifically detected by confocal microscopy following neutrophil ionomycin-induced degranulation. Fluorescence release was abolished in the presence of a selective hPR3 inhibitor, indicating that pro3-SBP is selectively cleaved by extracellular hPR3. Taken together, the present data support that pro3-SBP could be a convenient tool, allowing straightforward monitoring of human neutrophil activation.

A dark quencher genetically encodable voltage indicator (dqGEVI) exhibits high fidelity and speed

Voltage sensing with genetically expressed optical probes is highly desirable for large-scale recordings of neuronal activity and detection of localized voltage signals in single neurons. Most genetically encodable voltage indicators (GEVI) have drawbacks including slow response, low fluorescence, or excessive bleaching. Here we present a dark quencher GEVI approach (dqGEVI) using a Förster resonance energy transfer pair between a fluorophore glycosylphosphatidylinositol-enhanced green fluorescent protein (GPI-eGFP) on the outer surface of the neuronal membrane and an azo-benzene dye quencher (D3) that rapidly moves in the membrane driven by voltage. In contrast to previous probes, the sensor has a single photon bleaching time constant of ∼40 min, has a high temporal resolution and fidelity for detecting action potential firing at 100 Hz, resolves membrane de- and hyperpolarizations of a few millivolts, and has negligible effects on passive membrane properties or synaptic events. The dqGEVI approach should be a valuable tool for optical recordings of subcellular or population membrane potential changes in nerve cells.

The chromatographic behaviour of new double-labelled oligodeoxynucleotide probes containing azaphthalocyanine dye as a quencher with respect to evaluation of their purity.

The influence of experimental conditions on chromatographic behaviour of promising oligodeoxynucleotide double-labelled molecular probes containing an azaphthalocyanine macrocycle as a perspective dark quencher was studied. A recently introduced new stationary phase based on styrene-divinylbenzene copolymer was tested. The planar and hydrophobic structure of the azaphthalocyanine is considerably different from those of currently used fluorophores and quenchers. Thus, the most challenging issue was the separation of the double-labelled probe from its main impurity represented by a mono-labelled probe, containing only the azaphthalocyanine macrocycle. The absorbance measurement cannot simply determine this impurity, and its presence fundamentally compromises the biological assay. The commonly used gradient elution was not suitable and isocratic conditions seemed to be more appropriate. The azaphthalocyanine moiety influences the properties of the modified oligodeoxynucleotides substantially, and thus their chromatographic behaviour was determined predominantly by this quencher. Acetonitrile was the preferred organic solvent for the analysis of probes containing the azaphthalocyanine quencher and the effect of ion-pairing reagents was dependent on the probe structure. The temperature seemed to be an effective parameter for fine-tuning of the separation and mass transfer improvement. Generally, our findings could be helpful in method development for purity evaluation of double-labelled oligodeoxynucleotide probes and semipreparative methods.

Interaction of Cucurbit[7]uril With Protease Substrates: Application to Nanosecond Time-Resolved Fluorescence Assays.

We report the use of the macrocyclic host cucurbit[7]uril (CB7) as a supramolecular additive in nanosecond time-resolved fluorescence (Nano-TRF) assays for proteases to enhance the discrimination of substrates and products and, thereby, the sensitivity. A peptide substrate was labeled with 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) as a long-lived (>300 ns) fluorescent probe and 3-nitrotyrosine was established as a non-fluorescent fluorescence resonance energy transfer (FRET) acceptor that acts as a “dark quencher.” The substrate was cleaved by the model proteases trypsin and chymotrypsin and the effects of the addition of CB7 to the enzyme assay mixture were investigated in detail using UV/VIS absorption as well as steady-state and time-resolved fluorescence spectroscopy. This also allowed us to identify the DBO and nitrotyrosine residues as preferential binding sites for CB7 and suggested a hairpin conformation of the peptide, in which the guanidinium side chain of an arginine residue is additionally bound to a vacant ureido rim of one of the CB7 hosts.

Energy transfer from luminescent gold nanoclusters to non-luminescent silver nanoparticles from a new perspective

Histidine protected luminescent gold nanoclusters (Au@His NCs) were synthesized that interact with non-luminescent silver nanoparticles (Ag NPs) through a resonance energy transfer (RET) process where the latter acts as dark quencher. The interaction is collisional in nature as deduced by applying the Stern-Volmer equation. On external addition of a model transport protein (human serum albumin, HSA), the surface of the Au@His NCs accepts the proteins through physisorption that results into considerable enhancement of the luminescence of the Au@His NCs. The protein adsorbed Au@His NCs, on the other hand, possibly cling to the surface of the larger Ag NPs. This is confirmed from the quenching pattern of the emission from the Au@His NCs and time-resolved spectroscopy. The process contravenes the efficiency of RET and reduces the transfer of energy from the more luminescent Au@His NCs to the non-luminescent Ag NPs. On interaction with Ag NPs, the emission maximum shifts considerably to higher wavelength suggesting stabilization (due to restricted translational and rotational motions) of the Au@His NCs. The results are intriguing since the protein-aided adsorption of the luminescent Au@His NCs could restrict the energy transfer to the adsorbent (Ag NPs) in a quantitative way.

Synthesis and J-Dimer Formation of Tetrapyrazinoporphyrazines with Different Functional Groups for Potential Biomolecular Probe Applications.

Though tetrapyrazinoporphyrazines (TPyzPzs) are generally presented as universal dark quenchers for oligonucleotide probes, the availability of TPyzPzs bearing different functional groups suitable for attachment to 3', and 5' ends or intrastrand positions remains rather limited. Therefore, a synthetic route to hexa(bis(2-methoxyethyl)amino) or hexa(diethylamino) TPyzPzs functionalized by an azide, hydroxy, or carboxy group or their combinations was developed. Studies of self-assembly into J-dimers in nonpolar solvents and their stability upon titration with pyridine (association constants, KP values, ranging 0.32-12.7×102 M-1 ) revealed that smaller peripheral substituents and functionalization of TPyzPzs improves the stability of J-dimers. ΦΔ and ΦF were low for the monomers (ΦF <0.0001, ΦΔ <0.008, DMF) due to quenching by intramolecular charge transfer; however, they increased in nonpolar solvents and after self-assembly into J-dimer (up to ΦF =0.027, ΦΔ =0.28).

Fluorophore photostability and saturation in the hotspot of DNA origami nanoantennas

Fluorescent dyes used for single-molecule spectroscopy can undergo millions of excitation-emission cycles before photobleaching. Due to the upconcentration of light in a plasmonic hotspot, the conditions for fluorescent dyes are even more demanding in DNA origami nanoantennas. Here, we briefly review the current state of fluorophore stabilization for single-molecule imaging and reveal additional factors relevant in the context of plasmonic fluorescence enhancement. We show that despite the improved photostability of single-molecule fluorophores by DNA origami nanoantennas, their performance in the intense electric fields in plasmonic hotspots is still limited by the underlying photophysical processes, such as formation of dim states and photoisomerization. These photophysical processes limit the photon count rates, increase heterogeneity and aggravate quantification of fluorescence enhancement factors. These factors also reduce the time resolution that can be achieved in biophysical single-molecule experiments. Finally, we show how the photophysics of a DNA hairpin assay with a fluorophore-quencher pair can be influenced by plasmonic DNA origami nanoantennas leading to implications for their use in fluorescence-based diagnostic assays. Especially, we show that such assays can produce false positive results by premature photobleaching of the dark quencher.

Multiplexed non-invasive tumor imaging of glucose metabolism and receptor-ligand engagement using dark quencher FRET acceptor.

Rationale: Following an ever-increased focus on personalized medicine, there is a continuing need to develop preclinical molecular imaging modalities to guide the development and optimization of targeted therapies. Near-Infrared (NIR) Macroscopic Fluorescence Lifetime Förster Resonance Energy Transfer (MFLI-FRET) imaging offers a unique method to robustly quantify receptor-ligand engagement in live intact animals, which is critical to assess the delivery efficacy of therapeutics. However, to date, non-invasive imaging approaches that can simultaneously measure cellular drug delivery efficacy and metabolic response are lacking. A major challenge for the implementation of concurrent optical and MFLI-FRET in vivo whole-body preclinical imaging is the spectral crowding and cross-contamination between fluorescent probes.Methods: We report on a strategy that relies on a dark quencher enabling simultaneous assessment of receptor-ligand engagement and tumor metabolism in intact live mice. Several optical imaging approaches, such as in vitro NIR FLI microscopy (FLIM) and in vivo wide-field MFLI, were used to validate a novel donor-dark quencher FRET pair. IRDye 800CW 2-deoxyglucose (2-DG) imaging was multiplexed with MFLI-FRET of NIR-labeled transferrin FRET pair (Tf-AF700/Tf-QC-1) to monitor tumor metabolism and probe uptake in breast tumor xenografts in intact live nude mice. Immunohistochemistry was used to validate in vivo imaging results.Results: First, we establish that IRDye QC-1 (QC-1) is an effective NIR dark acceptor for the FRET-induced quenching of donor Alexa Fluor 700 (AF700). Second, we report on simultaneous in vivo imaging of the metabolic probe 2-DG and MFLI-FRET imaging of Tf-AF700/Tf-QC-1 uptake in tumors. Such multiplexed imaging revealed an inverse relationship between 2-DG uptake and Tf intracellular delivery, suggesting that 2-DG signal may predict the efficacy of intracellular targeted delivery.Conclusions: Overall, our methodology enables for the first time simultaneous non-invasive monitoring of intracellular drug delivery and metabolic response in preclinical studies.

A Phthalocyanine‐Based Glutathione‐Activated Photosensitizer with a Ferrocenyl Boron Dipyrromethene Dark Quencher for Photodynamic Therapy

AbstractThe front cover artwork is provided by the Dennis K. P. Ng group from the Chinese University of Hong Kong, and the Pui‐Chi Lo group from the City University of Hong Kong. The image illustrates the activation of a phthalocyanine‐based photosensitizer through cleavage of the disulfide linker by intracellular glutathione (GSH) and separation from the ferrocenyl boron dipyrromethene‐based dark quencher. Read the full text of the Article at 10.1002/cptc.201900115.

Construction of cathepsin B-responsive fluorescent probe and photosensitizer using a ferrocenyl boron dipyrromethene dark quencher

A ferrocenyl boron dipyrromethene (BODIPY) has been developed and utilized as a dark quencher to construct a cathepsin B-responsive fluorescent probe and photosensitizer. The smart fluorescent probe and photosensitizer (Pc-FcQ) contains a zinc(II) phthalocyanine as the fluorescent and photosensitizing unit which is conjugated to the ferrocenyl BODIPY dark quencher via a cathepsin B-cleavable peptide substrate [Gly-Phe-Leu-Gly-Lys]. The photosensitizing properties of Pc-FcQ, including fluorescence and singlet oxygen generation, are significantly quenched through energy transfer to the BODIPY unit and subsequently by the photoinduced electron transfer from the nearby ferrocenyl moiety. Upon exposure of cathepsin B in human hepatocellular carcinoma HepG cells, the fluorescence emission of Pc-FcQ could be restored, indicating the cleavage of the peptide substrate and the separation of the phthalocyanine and ferrocenyl BODIPY unit. However, the intracellular fluorescence intensity of Pc-FcQ was largely diminished after the cells were pre-treated with cathepsin B inhibitor. Its intracellular fluorescence intensity was comparable to that of the control compound in which the peptide substrate was replaced by the non-cleavable one [Gly-Gly-Gly-Gly-Lys]. The singlet oxygen generation of Pc-FcQ was also examined in HepG2 cells as reflected by the cytotoxicity assay. The Pc-FcQ exhibited higher potency when compared with the non-cleavable analogue due to the cleavage of peptide substrate and the detachment of the BODIPY dark quencher from the phthalocyanine. The activation of the Pc-FcQ was also demonstrated in tumor-bearing nude mice. After intratumoral injection of Pc-FcQ, the fluorescence intensity at the tumor region increased gradually over 10 h as a result of the detachment of the dark quencher upon the action of cathepsin B. All the results suggest that this ferrocenyl BODIPY could serve as an efficient dark quencher and the resulting Pc-FcQ could act as the cathepsin B-responsive fluorescent probe and activatable photosensitizer.