Optimizing protein crosslinking control: Synergistic quenching effects of glycine, histidine, and lysine on glutaraldehyde reactions.

Glutaraldehyde (GA) is a protein crosslinker widely used in biochemical and pharmaceutical research because it can rapidly stabilize and immobilize substrates via amine group interactions. However, controlling GA crosslinking is challenging owing to its swift reactivity and the influence of various solution conditions, such as pH and concentrations of the substrate and crosslinker. Although extensive research has focused on GA cross-linking mechanisms, studies on quenching, which is critical for preventing non-specific aggregation during prolonged storage, remain sparse. This study examines the quenching efficiency of a combined amino acid mixture of glycine, histidine, and lysine, which are commonly used as individual quenchers. Our findings, confirmed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis, demonstrate that this amino acid blend offers superior quenching compared to single amino acids, enhancing quenching activity across a wide pH spectrum. These results provide a novel approach for mitigating the high reactivity of GA with implications for improving sample preservation and stabilization in a range of biochemical applications, including microscopy and cell fixation.

Fluorescence Quenching and the Chamber of Nitroaromatics: A Dinaphthoylated Oxacalix[4]arene's (DNOC) Adventure Captured through Computational and Experimental Study.

This research presents the application of Dinaphthoylated Oxacalix[4]arene (DNOC) as a novel fluorescent receptor for the purpose of selectively detecting nitroaromatic compounds (NACs). The characterization of DNOC was conducted through the utilization of spectroscopic methods, including 1H-NMR, 13C-NMR, and ESI-MS. The receptor demonstrated significant selectivity in acetonitrile towards several nitroaromatic analytes, such as MNA, 2,4-DNT, 2,3-DNT, 1,3-DNB, 2,6-DNT, and 4-NT. This selectivity was validated by the measurement of emission spectra. The present study focuses on the examination of binding constants, employing Stern-Volmer analysis, as well as the determination of the lowest detection limit (3σ/Slope) and fluorescence quenching. These investigations aim to provide insights into the inclusion behavior of DNOC with each of the six analytes under fluorescence spectra investigation. Furthermore, the selectivity trend of the ligand DNOC for NAC detection is elucidated using Density Functional Theory (DFT) calculations conducted using the Gaussian 09 software. The examination of energy gaps existing between molecular orbitals, namely the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO), provides a valuable understanding of electron-transfer processes and electronic interactions. Smaller energy gaps are indicative of heightened selectivity resulting from favorable electron-transfer processes, whereas bigger gaps suggest less selectivity attributable to weaker electronic contacts. This work integrates experimental and computational methodologies to provide a full understanding of the selective binding behavior of DNOC. As a result, DNOC emerges as a viable chemical sensor for detecting nitroaromatic explosives.

ZnS quantum dots surface-loaded with zinc(II) ions as a viable fluorescent probe for glutathione.

The fluorescence of ZnS quantum dots in colloidal water solution changes very slightly on addition of glutathione (GSH) but is strongly enhanced in the presence of Zn(II) even in concentrations as low as 10 µM. The resulting Zn(II)-enhanced fluorescence is found to be quenched by GSH. In contrast to GSH, cysteine does not cause an effect. Response surface methodology was applied to optimize the experimental parameters. The best data can be obtained at 305/427 nm as excitation/emission wavelengths. These findings were used to design an indirect method for the fluorometric determination of GSH that has a 0.9 µM detection limit and a response that is linear in the 2.0-104.0 µM GSH concentration range. The relative standard deviation at a level of 65 µM of GSH (for n = 5) is 1.9%. Graphical abstract Schematic presentation of the detection strategy for glutathione and the influence of the mediator (Zn2+). Direct pathway shows that the glutathione cannot cause a change in the blue fluorescence of ZnS QDs. The presence of Zn2+ causes the enhancement of the fluorescence intensity, and this generates the indirect pathway to glutathione detection.

Ratiometric determination of hydrogen peroxide based on the size-dependent green and red fluorescence of CdTe quantum dots capped with 3-mercaptopropionic acid.

A ratiometric fluorescent nanoprobe is described for the detection of H2O2. It is based on the use of a mixture of green-emitting CdTe quantum dots (GQDs) and red-emitting CdTe QDs (RQDs). The two kinds of QDs have different size and different fluorescence response towards H2O2. The ratio of the emission intensities at 606 and 510 nm (under 365 nm photoexcitation) can be used as the analytical information. Even without any chemical modification of the surface of the QDs, the probe display high sensitivity and selectivity for H2O2. The fluorescence of small QDs is more effectively quenched by H2O2. Stern-Volmer analysis showed both static and dynamic quenching to occur. The probe works well in the 10~125 µM H2O2 concentration range and has a 0.3 µM detection limit (3σ/slope). Graphical abstract Schematic presentation of the ratiometric fluorescent nanoprobe composed of green-emitting and red-emitting CdTe QDs. λ, I, and k are the emission wavelength, emission intensity, and quenching/enhancement coefficient, respectively. The subscript 0 and 1 present the green-emitting and red-emitting CdTe QDs, respectively.

Fluorometric determination of RNase H via a DNAzyme conjugated to reduced graphene oxide, and its application to screening for inhibitors and activators.

A new fluorometric method is delineated for the detection of RNase H activity by combining DNAzyme with reduced graphene oxide (rGO). In the absence of RNase H, the fluorescence of FAM-labeled probe is quenched due to the strong adsorption on the rGO. The presence of RNase H can release the active DNAzyme from the DNA-RNA chimeric strand. This triggers the cleavage of the signal probe at the rA site with the help of the cofactor Mg2+. The recycle cleavage can directly result in the amplified signal emitted by the FAM-labeled short fragment. The method allows the activity of RNase H to be detected in a linear range of 0.01 to 5 U·mL-1. The detection limit of 0.018 U·mL-1 is calculated by the principle of three-time standard deviation over the blank signal. Then, RNase H-targeting natural compounds were screened for their inhibitory action. Among the investigated compounds, five were screened as RNase H inhibitors in a concentration-dependent manner, and 4 compounds were identified as activators. Finally, the method was reliably used for discriminating the difference of RNase H activity in human serum. It is found that RNase H activity was upregulated in patients with hepatitis C virus infection. Graphical abstract The schematic presentation of rGO-DNAzyme-based RNase H detection. RNase H triggers the active DNAzyme releasing from the DNA-RNA chimeric strand, which can cleavage probes to FAM-labeled short fragments and make the fluorescence signal cycle amplified.

Ratiometric fluorometric determination of silver(I) by using blue-emitting silicon- and nitrogen-doped carbon quantum dots and red-emitting N-acetyl-L-cysteine-capped CdTe quantum dots.

A ratiometric fluorometric assay for silver(I) is described. The method makes use of a dually emitting quantum dot hybrid, which is composed of (a) blue-fluorescent silicon- and nitrogen-doped carbon quantum dots (CQDs), and (b) of red-emitting CdTe quantum dots (QDs) capped with N-acetyl-L-cysteine. The red-emitting CdTe QDs undergo strong and specific quenching by Ag(I), whereas the blue-emitting N,Si-CQDs are not quenched. The two kinds of QDs are mixed and used as a ratiometric fluorescent probe. A linear relationship is found between the log of intensities [(I608/I441)0/(I608/I441)] and the concentration of Ag(I) in the range from 5.0-1000 nM, and the limit of detection (at S/N = 3) is 1.7 nM. Possible interferents (including 17 general metal ions, 12 anions and fulvic acid) do not interfere with the determination. The assay was successfully used for the determination of Ag(I) in surface water and wastewater samples. The fluorescence quenching mechanism of the ratiometric assay system was also discussed in detailed. Graphical abstract Schematic representation of a ratiometric probe composed of silicon- and nitrogen-doped carbon quantum dots (N,Si-CQDs) and CdTe quantum dots capped by N-acetyl-L-cysteine (CdTe QDs). This dual-emission QDs hybrid was fabricated for ultrasensitive and highly selective detection of silver(I) in water samples.

Nanometal surface energy transfer-based lateral flow immunoassay for T2 toxin detection.

In this study, we incorporated nanometal surface energy transfer (NSET) in lateral flow immunoassay (LFIA) and explored the relationship between fluorescence quenching efficiency and detection sensitivity to improve sensitivity of NSET-LFIA system. We developed nine gold nanoparticles (GNPs) with absorption spectrum in the range of 520-605 nm as acceptors and quantum dot microspheres (QDMs) with emission spectrum of 530, 570, and 610 nm as donors. By analyzing the overlap integral area, fluorescence quenching efficiency, and detection sensitivity of 27 donor-acceptor pairs, we observed that the larger overlap integral area led to higher fluorescence quenching efficiency and detection sensitivity. A maximum fluorescence quenching efficiency of 91.0% was obtained from the combination of GNPs at 605 nm and QDMs at 610 nm, achieving the highest detection sensitivity. We developed NSET-LFIA for the detection of T2 toxin with a limit of detection of 0.04 ng/mL, which was 10-times higher than that obtained via conventional GNP-LFIA. NSET-LFIA represents a versatile, ultrasensitive and valuable screening tool for small molecules in real samples.

Dual-Modality Imaging with a Zwitterionic Fluorescent Probe for Reversible Monitoring of Mitochondrial Membrane Potential Dynamics.

Traditional fluorescence intensity-based probes face challenges in accurately measuring mitochondrial membrane potential (MMP) due to intramolecular fluorescence quenching. In this work, we introduce a novel approach by incorporating quenching moieties within the zwitterionic probe to eliminate self-quenching interference, thus, enabling real-time and precise visualization of reversible MMP changes. We synthesized a zwitterionic fluorescent probe consisting of silicon-rhodamine (SiR) that was hydroxyl-substituted on the bay position of perylene diimides (PDIs) connected via a polyethylene glycol (PEG) linker. The lipophilic cationic SiR facilitates the entry of the PDI into the mitochondria, where the alkaline pH environment (pH = 8.0) ionizes the hydroxyl to a negatively charged species, affecting the quenching efficiency of SiR depending on the distance between the PDI and SiR moieties regulated by the MMP. The rigid aromatic ring of the PDI and strong hydrophobic interactions with the lipid bilayer, along with the inhibitory effect of the negatively charged hydroxyl on internalization, ensure the retention of PDI within the mitochondria. As the MMP decreases, SiR shifts outward, reducing quenching by phenolic anions and restoring fluorescence. Conversely, as the MMP increases, SiR moves inward, intensifying quenching by phenolic ions and reducing fluorescence, enabling reversible visualization monitoring of the MMP. This strategy overcomes the limitations of traditional intensity-based probes, providing a new avenue for reversible monitoring of the MMP.

Coordination Polymers as a Functional Material for the Selective Molecular Recognition of Nitroaromatics and ipso-Hydroxylation of Arylboronic Acids.

We report the synthesis and structural characterization of two coordination polymers (CPs), namely; [{Zn(L)(DMF)4 } ⋅ 2BF4 ]α (1) and [{Cd(L)2 (Cl)2 } ⋅ 2H2 O]α (2) (where L=N2 ,N6 -di(pyridin-4-yl)naphthalene-2,6-dicarboxamide). Crystal packing of 1 reveals the existence of channels running along the b- and c-axis filled by the ligated DMF and lattice anions, respectively. Whereas, crystal packing of 2 reveals that the metallacycles of each 1D chain are intercalating into the groove of adjacent metallacycles resulting in the stacking of 1D loop-chains to form a sheet-like architecture. In addition, both 1 and 2 were exploited as multifunctional materials for the detection of nitroaromatic compounds (NACs) as well as a catalyst in the ipso-hydroxylation of aryl/heteroarylboronic acids. Remarkably, 1 and 2 showed high fluorescence stability in an aqueous medium and displayed a maximum 88% and 97% quenching efficiency for 4-NPH, respectively among all the investigated NACs. The mechanistic investigation of NACs recognition suggested that the fluorescence quenching occurred via electron as well as energy transfer process. Furthermore, the ipso-hydroxylation of aryl/heteroarylboronic acids in presence of 1 and 2 gave up to 99% desired product yield within 15 min in our established protocol. In both cases, 1 and 2 are recyclable upto five cycles without any significant loss in their efficiency.

Nanostructure and Corresponding Quenching Efficiency of Fluorescent DNA Probes.

Based on the fluorescence resonance energy transfer (FRET) mechanism, fluorescent DNA probes were prepared with a novel DNA hairpin template method, with SiO2 coated CdTe (CdTe/SiO2) core/shell nanoparticles used as the fluorescence energy donors and gold (Au) nanoparticles (AuNPs) as the energy acceptors. The nanostructure and energy donor/acceptor ratio in a probe were controlled with this method. The relationship between the nanostructure of the probes and FRET efficiency (quenching efficiency) were investigated. The results indicated that when the donor/acceptor ratios were 2:1, 1:1, and 1:2; the corresponding FRET efficiencies were about 33.6%, 57.5%, and 74.2%, respectively. The detection results indicated that the fluorescent recovery efficiency of the detecting system was linear when the concentration of the target DNA was about 0.0446-2.230 nmol/L. Moreover, the probes showed good sensitivity and stability in different buffer conditions with a low detection limit of about 0.106 nmol/L.