Sequence Dependence of Fluorescent Quenching of Chromophores Covalently Bonded to Oligo-DNAs before and after Duplex Formation

The labeling of oligo- and polynucleotides with fluorescent probes is an important technique for the analysis of DNAs and RNAs. The effect of duplex formation with complementary oligo-DNA on the quenching behavior of two fluorescent chromophores (eosin, Eo and tetramethylrhodamine, TMR) attached to the 5′-terminal of various 10mer oligo-DNAs was investigated and the dependence of the quenching on DNA base sequence is discussed. We found that guanine residues played a major role in the quenching of the fluorescence of the chromophores. Guanine residues on the complementary DNA near the chromophores, in particular, had a significant influence on the quenching.     

An ultrasensitive quantum dots fluorescent polarization immunoassay based on the antibody modified Au nanoparticles amplifying for the detection of adenosine triphosphate

In this work, an ultrasensitive fluorescent polarization immunoassay (FPIA) method based on the quantum dot/aptamer/antibody/gold nanoparticles ensemble has been developed for the detection of adenosine triphosphate (ATP). DNA hybridization is formed when ATP is present in the PBS solution containing the DNA-conjugated quantum dots (QDs) and antibody-AuNPs. The substantial sensitivity improvement of the antibody-AuNPs-enhanced method is mainly attributed to the slower rotation of fluorescent unit when QDs-labeled oligonucleotides hybridize with antibody modified the gold nanoparticle. As a result, the fluorescent polarization (FP) values of the system increase significantly. Under the optimal conditions, a linear response with ATP concentration is ranged from 8 × 10⁻¹² M to 2.40 × 10⁻⁴ M. The detection limit reached as low as 1.8 pM. The developed work provides a sensitive and selective immunoassay protocol for ATP detection, which could be applied in more bioanalytical systems.     

Synthesis and characterization of new selective Zn fluorescent probes for functionalization: in vitro Cell imaging applications

Herein the synthesis and characterization of new, lipophilic highly Zn²⁺-selective fluorescent probes are reported. High affinity for zinc (K_d 1.1–8.0 nM) over other biologically relevant metals and mixtures of metals was observed. Excitation at 360 nm afforded an emission spectrum with maximum at 530 nm for the zinc bound complex. The linear relationship between fluorescence intensity and zinc concentration indicates that FZnA-probes can be used for quantification. The probes have been synthesized in 28–45% overall yield and the feasibility for further functionalization with biologically relevant side chains has been demonstrated. In vitro studies using PC12 cells and 10 μM of one of the novel probes (FZnA-Ada) visualized endogenous labile Zn²⁺ after 45 min incubation time.     

Fluorescent staining of protein in SDS polyacrylamide gels by salicylaldehyde azine

As a noncovalent fluorescence probe, in this study, salicylaldehyde azine (SA) was introduced as a sensitive fluorescence‐based dye for detecting proteins both in 1D and 2D polyacrylamide electrophoresis gels. Down to 0.2 ng of single protein band could be detected within 1 h, which is similar to that of glutaraldehyde‐silver stain, but approximately four times higher than that of SYPRO Ruby fluorescent stain. Furthermore, comparative analysis of the MS compatibility of SA stain with SYPRO Ruby stain indicated that SA stain is compatible with the downstream of protein identification by LC‐MS/MS. Additionally, the probable mechanism of the SA stain was investigated by molecular docking. The results demonstrated that the interaction between SA and protein was mainly contributed by hydrogen bonding and hydrophobic forces.     

Benign synthesis of N-(8-quinolyl)pyridine-2-carboxamide) ligand (Hbpq), and its Ni (II) and Cu (II) complexes. A fluorescent probe for direct detection of nitric oxide in acetonitrile solution based on Hbpq copper(II) acetate interaction

The ligand Hbpq = N-(8-quinolyl)pyridine-2-carboxamide) has been prepared using tetrabutylammonium bromide (TBAB) as an environmentally friendly reaction medium. Four new complexes of this ligand, [M(bpq)X] (M = Cu(II), X = SCN̄ (1), N₃̄ (2); M = Ni(II), X = SCN̄ (3), N₃̄ (4)), have also been synthesized and fully characterized. The crystal and molecular structures of [Cu(bpq)(NCS)]_n (1) have been determined by X-ray crystallography. Copper(II) ion adopts a distorted square pyramidal (4 + 1) coordination in this complex. Hbpq ligand shows a strong emission at 500 nm in acetonitrile solution. The emission is quenched in the presence of copper(II) acetate, apparently because of the formation of [Cu(L)(OAc)(H₂O)] complex. Introduction of nitric oxide (NO) into the acetonitrile solution at room temperature induces an increase in the fluorescence intensity, presumably due to the reduction of Cu(II) to Cu(I). This process is reversible and can form a basis for direct detection of NO.     

A novel colorimetric and “turn-on” fluorescent sensor for selective detection of Cu2+

In this work, a new highly selective and sensitive fluorescent sensor for detecting Cu²⁺ was developed based on rhodamine fluorophore. It displayed strong fluorescence “turn-on” effect upon addition of Cu²⁺, and possessed the function of naked eye recognition. The fluorescence enhancement also enabled the sensor to quantitatively analyze Cu²⁺ due to the formation of a stable 1:1 metal–ligand complex in a short time, and the complex possesses relatively good pH stability. In addition, the density functional theory calculations were adopted to investigate the molecular orbitals as well as the spatial structure. Simultaneously, the cell imaging and zebra fish experiments provided a broader application prospect in biological system.     

Improved Stabilities of Labeling Probes for the Selective Modification of Endogenous Proteins in Living Cells and In Vivo

To date, various affinity-based protein labeling probes have been developed and applied in biological research to modify endogenous proteins in cell lysates and on the cell surface. However, the reactive groups on the labeling probes are also the cause of probe instability and nonselective labeling in a more complex environment, e. g., intracellular and in vivo. Here, we show that labeling probes composed of a sterically stabilized difluorophenyl pivalate can achieve efficient and selective labeling of endogenous proteins on the cell surface, inside living cells and in vivo. As compared with the existing protein labeling probes, probes with the difluorophenyl pivalate exhibit several advantages, including long-term stability in stock solutions, resistance to enzymatic hydrolysis and can be customized easily with diverse fluorophores and protein ligands. With this probe design, endogenous hypoxia biomarker in living cells and nude mice were successfully labeled and validated by in vivo, ex vivo, and immunohistochemistry imaging.     

Monitoring intracellular pH fluctuation with an excited-state intramolecular proton transfer-based ratiometric fluorescent sensor

Intracellular pH is a key parameter related to various biological and pathological processes. In this study, a ratiometric pH fluorescent sensor ABTT was developed harnessing the amino-type excited-state intramolecular proton transfer (ESIPT) process. Relying on whether the ESIPT proceeds normally or not, ABTT exhibited the yellow fluorescence in acidic media, or cyan fluorescence in basic condition. According to the variation, ABTT behaved as a promising sensor which possessed fast and reversible response to pH change without interference from the biological substances, and exported a steady ratiometric signal (I₄₇₈/I₅₄₆). Moreover, due to the ESIPT effect, large Stokes shift and high quantum yield were also exhibited in ABTT. Furthermore, ABTT was applied for monitoring the pH changes in living cells and visualizing the pH fluctuations under oxidative stress successfully. These results elucidated great potential of ABTT in understanding pH-dependent physiological and pathological processes.     

Blue emission from InGaN/GaN hexagonal pyramid structures

Cathodoluminescence (CL) from InGaN grown on GaN hexagonal pyramid structures has been investigated. The facet structure can be controlled by the growth temperature and reactor pressure. GaN pyramid structures surrounded with facets were grown at 1020 C at a pressure of 500 Torr by low-pressure metalorganic vapor phase epitaxy (LP-MOVPE). The indium mole fraction in the InGaN film depends on the facet structure. The thickness of the InGaN and the peak wavelength and intensity of the CL from the InGaN gradually increased from the bottom to the top of the facets.     

Solvent and pH Effects on the Fluorescence of 7-(Dimethylamino)-2-Fluorenesulfonate

A novel water-soluble solvatochromic molecule, 7-(dimethylamino)-2-fluorenesulfonate (2,7-DAFS), was prepared by a three-step reaction from 2-nitrofluorene in good overall yield. The pH and solvent effects on the UV-VIS absorption and fluorescence spectra of 2,7-DAFS have been studied. Protonation of the dimethylamino group switches the absorption from intramolecular charge-transfer (ICT) to π → π* transition. The ground state pKa value of 2,7-DAFS was determined as 4.51. The fluorescence spectrum of the excited basic form, *(DAFS), shows a structureless single band with a large Stokes shift, whereas that of the acidic form, *(⁺HDAFS), exhibits a structured band with a small Stokes shift. The emission intensities of the basic and acidic forms versus pH/Ho plots show stretched sigmoidal curves and indicate that (1) the rate of deprotonation of *(⁺HDAFS) is comparable to the fluorescence decay of the species, and (2) the efficient proton-induced quenching of *(DAFS) fluorescence occurs. The pKa* was estimated as −1.7 from the fluorescence titration curve. The fluorescence maximum of *(DAFS) is blue-shifted as the polarity of solvent decreases. However, no clear dependency of the emission intensity and spectral half width, and thus fluorescence quantum yield, on the solvent polarity was revealed. It appears that the fluorescence sensitivity of 2,7-DAFS is 15 ∼ 25 times greater than the sensitivity of a widely utilized fluorescent probe, 5-(dimethylamino)-1-naphthalenesulfonate. This higher sensitivity, together with the ease of derivatization, would provide the fluorene-based fluorescent molecules significant advantages for a variety of applications.