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16 pages, 4269 KiB

Open AccessArticle

Multiplexed In Situ Protein Profiling with High-Performance Cleavable Fluorescent Tyramide

by Thai Pham, Renjie Liao, Joshua Labaer and Jia Guo

Molecules 2021, 26(8), 2206; https://doi.org/10.3390/molecules26082206 - 12 Apr 2021

Cited by 6 | Viewed by 2956

Understanding the composition, function and regulation of complex cellular systems requires tools that quantify the expression of multiple proteins at their native cellular context. Here, we report a highly sensitive and accurate protein in situ profiling approach using off-the-shelf antibodies and cleavable fluorescent [...] Read more.

Understanding the composition, function and regulation of complex cellular systems requires tools that quantify the expression of multiple proteins at their native cellular context. Here, we report a highly sensitive and accurate protein in situ profiling approach using off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this method, protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and CFT. Subsequently, the fluorophores are efficiently cleaved by mild chemical reagents, which simultaneously deactivate HRP. Through reiterative cycles of protein staining, fluorescence imaging, fluorophore cleavage, and HRP deactivation, multiplexed protein quantification in single cells in situ can be achieved. We designed and synthesized the high-performance CFT, and demonstrated that over 95% of the staining signals can be erased by mild chemical reagents while preserving the integrity of the epitopes on protein targets. Applying this method, we explored the protein expression heterogeneity and correlation in a group of genetically identical cells. With the high signal removal efficiency, this approach also enables us to accurately profile proteins in formalin-fixed paraffin-embedded (FFPE) tissues in the order of low to high and also high to low expression levels. Full article

(This article belongs to the Special Issue Fluorescence Spectroscopy of Biomolecules)

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12 pages, 3454 KiB

Open AccessArticle

Revisiting the Rate-Limiting Step of the ANS–Protein Binding at the Protein Surface and Inside the Hydrophobic Cavity

by Chikashi Ota, Shun-ichi Tanaka and Kazufumi Takano

Molecules 2021, 26(2), 420; https://doi.org/10.3390/molecules26020420 - 14 Jan 2021

Cited by 34 | Viewed by 5727

Abstract

8-Anilino-1-naphthalenesulfonic acid (ANS) is used as a hydrophobic fluorescence probe due to its high intensity in hydrophobic environments, and also as a microenvironment probe because of its unique ability to exhibit peak shift and intensity change depending on the surrounding solvent environment. The [...] Read more.

8-Anilino-1-naphthalenesulfonic acid (ANS) is used as a hydrophobic fluorescence probe due to its high intensity in hydrophobic environments, and also as a microenvironment probe because of its unique ability to exhibit peak shift and intensity change depending on the surrounding solvent environment. The difference in fluorescence can not only be caused by the microenvironment but can also be affected by the binding affinity, which is represented by the binding constant (K). However, the overall binding process considering the binding constant is not fully understood, which requires the ANS fluorescence binding mechanism to be examined. In this study, to reveal the rate-limiting step of the ANS–protein binding process, protein concentration-dependent measurements of the ANS fluorescence of lysozyme and bovine serum albumin were performed, and the binding constants were analyzed. The results suggest that the main factor of the binding process is the microenvironment at the binding site, which restricts the attached ANS molecule, rather than the attractive diffusion-limited association. The molecular mechanism of ANS–protein binding will help us to interpret the molecular motions of ANS molecules at the binding site in detail, especially with respect to an equilibrium perspective. Full article

(This article belongs to the Special Issue Fluorescence Spectroscopy of Biomolecules)

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13 pages, 2260 KiB

Open AccessArticle

Nile-Red-Based Fluorescence Probe for Selective Detection of Biothiols, Computational Study, and Application in Cell Imaging

by Xiang Rong, Zhong-Yong Xu, Jin-Wu Yan, Zhi-Zhong Meng, Bin Zhu and Lei Zhang

Molecules 2020, 25(20), 4718; https://doi.org/10.3390/molecules25204718 - 14 Oct 2020

Cited by 10 | Viewed by 6256

Abstract

A new colorimetric and fluorescence probe NRSH based on Nile-red chromophore for the detection of biothiols has been developed, exhibiting high selectivity towards biothiols over other interfering species. NRSH shows a blue shift in absorption peak upon reacting with biothiols, from 587 nm [...] Read more.

A new colorimetric and fluorescence probe NRSH based on Nile-red chromophore for the detection of biothiols has been developed, exhibiting high selectivity towards biothiols over other interfering species. NRSH shows a blue shift in absorption peak upon reacting with biothiols, from 587 nm to 567 nm, which induces an obvious color change from blue to pink and exhibits a 35-fold fluorescence enhancement at 645 nm in red emission range. NRSH displays rapid (<1 min) response for H₂S, which is faster than other biothiols (>5 min). The detection limits of probe NRSH towards biothiols are very low (22.05 nM for H₂S, 34.04 nM for Cys, 107.28 nM for GSH and 113.65 nM for Hcy). Furthermore, NRSH is low cytotoxic and can be successfully applied as a bioimaging tool for real-time monitoring biothiols in HeLa cells. In addition, fluorescence mechanism of probe NRSH is further understood by theoretical calculations. Full article

(This article belongs to the Special Issue Fluorescence Spectroscopy of Biomolecules)

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11 pages, 2565 KiB

Open AccessArticle

Excited-State Proton Transfer in 8-Azapurines I: A Kinetic Analysis of 8-Azaxanthine Fluorescence

by Jacek Wierzchowski and Bogdan Smyk

Molecules 2020, 25(12), 2740; https://doi.org/10.3390/molecules25122740 - 12 Jun 2020

Cited by 7 | Viewed by 2464

Abstract

A super-continuum white laser with a half-pulse width of ~75 ps was used to observe the kinetics of a postulated excited-state proton transfer in 8-azaxanthine and its 8-methyl derivative. Both compounds exhibited dual emissions in weakly acidified alcoholic media, but only one band [...] Read more.

A super-continuum white laser with a half-pulse width of ~75 ps was used to observe the kinetics of a postulated excited-state proton transfer in 8-azaxanthine and its 8-methyl derivative. Both compounds exhibited dual emissions in weakly acidified alcoholic media, but only one band was present in aqueous solutions, exhibiting an abnormal Stokes shift (>12,000 cm⁻¹). It was shown that long-wavelength emissions were delayed relative to the excitation pulse within alcoholic media. The rise time was calculated to be 0.4–0.5 ns in both methanol and deuterated methanol. This is equal to the main component of the fluorescence decay in the short-wavelength band (340 nm). Time-resolved emission spectra (TRES) indicated a two-state photo-transformation model in both compounds. Global analysis of the time dependence revealed three exponential components in each compound, one of which had an identical rise-time, with the second attributed to a long-wavelength band decay (6.4 ns for aza-xanthine and 8.3 ns for its 8-methyl derivative). The origin of the third, intermediate decay time (1.41 ns for aza-xanthine and 0.87 ns for 8-methyl-azaxanthine) is uncertain, but decay-associated spectra (DAS) containing both bands suggest the participation of a contact ion pair. These results confirm the model of phototautomerism proposed earlier, but the question of the anomalous isotope effect remains unsolved. Full article

(This article belongs to the Special Issue Fluorescence Spectroscopy of Biomolecules)

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19 pages, 2694 KiB

Open AccessArticle

Excited State Dynamics of 8-Vinyldeoxyguanosine in Aqueous Solution Studied by Time-Resolved Fluorescence Spectroscopy and Quantum Mechanical Calculations

by Lara Martinez-Fernandez, Thomas Gustavsson, Ulf Diederichsen and Roberto Improta

Molecules 2020, 25(4), 824; https://doi.org/10.3390/molecules25040824 - 13 Feb 2020

Cited by 5 | Viewed by 2973

Abstract

The fluorescent base guanine analog, 8-vinyl-deoxyguanosine (8vdG), is studied in solution using a combination of optical spectroscopies, notably femtosecond fluorescence upconversion and quantum chemical calculations, based on time-dependent density functional theory (TD-DFT) and including solvent effect by using a mixed discrete-continuum model. In [...] Read more.

The fluorescent base guanine analog, 8-vinyl-deoxyguanosine (8vdG), is studied in solution using a combination of optical spectroscopies, notably femtosecond fluorescence upconversion and quantum chemical calculations, based on time-dependent density functional theory (TD-DFT) and including solvent effect by using a mixed discrete-continuum model. In all investigated solvents, the fluorescence is very long lived (3–4 ns), emanating from a stable excited state minimum with pronounced intramolecular charge-transfer character. The main non-radiative decay channel features a sizeable energy barrier and it is affected by the polarity and the H-bonding properties of the solvent. Calculations provide a picture of dynamical solvation effects fully consistent with the experimental results and show that the photophysical properties of 8vdG are modulated by the orientation of the vinyl group with respect to the purine ring, which in turn depends on the solvent. These findings may have importance for the understanding of the fluorescence properties of 8vdG when incorporated in a DNA helix. Full article

(This article belongs to the Special Issue Fluorescence Spectroscopy of Biomolecules)

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16 pages, 2466 KiB

Open AccessArticle

Excited State Lifetimes of Sulfur-Substituted DNA and RNA Monomers Probed Using the Femtosecond Fluorescence Up-Conversion Technique

by Matthew M. Brister, Thomas Gustavsson and Carlos E. Crespo-Hernández

Molecules 2020, 25(3), 584; https://doi.org/10.3390/molecules25030584 - 29 Jan 2020

Cited by 9 | Viewed by 4284

Abstract

Sulfur-substituted DNA and RNA nucleobase derivatives (a.k.a., thiobases) are an important family of biomolecules. They are used as prodrugs and as chemotherapeutic agents in medical settings, and as photocrosslinker molecules in structural-biology applications. Remarkably, excitation of thiobases with ultraviolet to near-visible light results [...] Read more.

Sulfur-substituted DNA and RNA nucleobase derivatives (a.k.a., thiobases) are an important family of biomolecules. They are used as prodrugs and as chemotherapeutic agents in medical settings, and as photocrosslinker molecules in structural-biology applications. Remarkably, excitation of thiobases with ultraviolet to near-visible light results in the population of long-lived and reactive triplet states on a time scale of hundreds of femtoseconds and with near-unity yields. This efficient nonradiative decay pathway explains the vanishingly small fluorescence yields reported for the thiobases and the scarcity of fluorescence lifetimes in the literature. In this study, we report fluorescence lifetimes for twelve thiobase derivatives, both in aqueous solution at physiological pH and in acetonitrile. Excitation is performed at 267 and 362 nm, while fluorescence emission is detected at 380, 425, 450, 525, or 532 nm. All the investigated thiobases reveal fluorescence lifetimes that decay in a few hundreds of femtoseconds and with magnitudes that depend and are sensitive to the position and degree of sulfur-atom substitution and on the solvent environment. Interestingly, however, three thiopyrimidine derivatives (i.e., 2-thiocytidine, 2-thiouridine, and 4-thiothymidine) also exhibit a small amplitude fluorescence component of a few picoseconds in aqueous solution. Furthermore, the N-glycosylation of thiobases to form DNA or RNA nucleoside analogues is demonstrated as affecting their fluorescence lifetimes. In aqueous solution, the fluorescence decay signals exciting at 267 nm are equal or slower than those collected exciting at 362 nm. In acetonitrile, however, the fluorescence decay signals recorded upon 267 nm excitation are, in all cases, faster than those measured exciting at 362 nm. A comparison to the literature values show that, while both the DNA and RNA nucleobase and thiobase derivatives exhibit sub-picosecond fluorescence lifetimes, the ¹ππ* excited-state population in the nucleobase monomers primarily decay back to the ground state, whereas it predominantly populates long-lived and reactive triplet states in thiobase monomers. Full article

(This article belongs to the Special Issue Fluorescence Spectroscopy of Biomolecules)

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11 pages, 2377 KiB

Open AccessArticle

A Ratiometric and Colorimetric Hemicyanine Fluorescent Probe for Detection of SO₂ Derivatives and Its Applications in Bioimaging

by Yan-Hong Qin, Xiao-Yi Jiang, Yuan-Fang Que, Jing-Yi Gu, Tong Wu, Ayinazhaer Aihemaiti, Ke-Xin Shi, Wen-Yu Kang, Bi-Ying Hu, Jin-Shuai Lan, Yue Ding and Tong Zhang

Molecules 2019, 24(21), 4011; https://doi.org/10.3390/molecules24214011 - 5 Nov 2019

Cited by 16 | Viewed by 4018

Abstract

Based upon the intramolecular charge transfer (ICT) mechanism, a novel ratiometric fluorescent probe EB was developed to detect SO₃²⁻/HSO₃⁻. The probe displayed both colorimetric and ratiometric responses toward SO₃²⁻/HSO₃⁻. It displayed [...] Read more.

Based upon the intramolecular charge transfer (ICT) mechanism, a novel ratiometric fluorescent probe EB was developed to detect SO₃²⁻/HSO₃⁻. The probe displayed both colorimetric and ratiometric responses toward SO₃²⁻/HSO₃⁻. It displayed a quick response (within 60 s), good selectivity and high sensitivity (a detection limit of 28 nM) towards SO₃²⁻/HSO₃⁻. The SO₃²⁻/HSO₃⁻ sensing mechanism was confirmed as the Michael addition reaction by ESI-MS. Moreover, the probe could be applied to measure the level of sulfite in real samples, like sugar and chrysanthemum, and it could also be used to detect SO₃²⁻/HSO₃⁻ in HepG2 cells through confocal fluorescence microscopy, which proved its practical application in clinical diagnosis. Full article

(This article belongs to the Special Issue Fluorescence Spectroscopy of Biomolecules)

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10 pages, 2338 KiB

Open AccessArticle

A Green-emitting Fluorescent Probe Based on a Benzothiazole Derivative for Imaging Biothiols in Living Cells

by Xiaohua Ma, Yuanqiang Hao, Jiaxiang Liu, Guoguang Wu and Lin Liu

Molecules 2019, 24(3), 411; https://doi.org/10.3390/molecules24030411 - 23 Jan 2019

Cited by 13 | Viewed by 3556

Abstract

A new green-emitting fluorescent probe 1 was developed for biothiol detection. The sensing mechanism was considered to be biothiol-induced cleavage of the 2,4-dinitrobenzene- sulfonate group in probe 1 and resulting inhibition of the probe’s photoinduced electron transfer (PET) process. Probe 1 exhibited favorable [...] Read more.

A new green-emitting fluorescent probe 1 was developed for biothiol detection. The sensing mechanism was considered to be biothiol-induced cleavage of the 2,4-dinitrobenzene- sulfonate group in probe 1 and resulting inhibition of the probe’s photoinduced electron transfer (PET) process. Probe 1 exhibited favorable properties such as excellent selectivity, highly sensitive (0.12 µM), large Stokes shift (117 nm) and a remarkable turn-on fluorescence signal (148-fold). Furthermore, confocal fluorescence imaging indicated that probe 1 was membrane-permeable and suitable for visualization of biothiols in living A549 cells. Full article

(This article belongs to the Special Issue Fluorescence Spectroscopy of Biomolecules)

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