Molecular Quenchers Research Articles

A Fluorescence Resonance Energy Transfer Aptasensor for Aflatoxin B1 Based on Ligand-Induced ssDNA Displacement

In this study, a fluorescence resonance energy transfer (FRET)-based aptasensor for the detection of aflatoxin B1 (AFB1) was designed using a carboxyfluorescein (FAM)-labeled aptamer and short complementary DNA (cDNA) labeled with low molecular quencher RTQ1. The sensing principle was based on the detection of restored FAM-aptamer fluorescence due to the ligand-induced displacement of cDNA in the presence of AFB1, leading to the destruction of the aptamer/cDNA duplex and preventing the convergence of FAM and RTQ1 at the effective FRET distance. Under optimal sensing conditions, a linear correlation was obtained between the fluorescence intensity of the FAM-aptamer and the AFB1 concentration in the range of 2.5–208.3 ng/mL with the detection limit of the assay equal to 0.2 ng/mL. The assay time was 30 min. The proposed FRET aptasensor has been successfully validated by analyzing white wine and corn flour samples, with recovery ranging from 76.7% to 91.9% and 84.0% to 86.5%, respectively. This work demonstrates the possibilities of labeled cDNA as an effective and easily accessible tool for sensitive AFB1 detection. The homogeneous FRET aptasensor is an appropriate choice for contaminant screening in complex matrices.

Uncovering Molecular Quencher Effects on FRET Phenomena in Microsphere-Immobilized Probe Systems.

Double-stranded (ds) oligonucleotide probes composed of quencher-dye sequence pairs outperform analogous single-stranded (ss) probes due to their superior target sequence specificity without any prerequisite target labeling. Optimizing sequence combinations for dsprobe design requires promoting a fast, accurate response to a specific target sequence while minimizing spontaneous dsprobe dissociation events. Here, flow cytometry is used to rapidly interrogate the stability and selective responsiveness of 20 candidate LNA and DNA dsprobes to a 24 base-long segment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and ∼243 degenerate RNA sequences serving as model variants. Importantly, in contrast to quantifying binding events of dye-labeled targets via flow cytometry, the current work employs the Förster resonance energy transfer (FRET)-based detection of unlabeled RNA targets. One DNA dsprobe with a 15-base-long hybridization partner containing a central abasic site emerged as very stable yet responsive only to the SARS-CoV-2 RNA segment. Separate displacement experiments, however, indicated that ∼12% of these quencher-capped hybridization partners remain bound, even in the presence of an excess SARS-CoV-2 RNA target. To examine their quenching range, additional titration studies varied the ratios and spatial placement of nonquencher and quencher-capped hybridization partners in the dsprobes. These titration studies indicate that these residual, bound quencher-capped partners, even at low percentages, act as nodes, enabling both static quenching effects within each residual dsprobe as well as longer-range quenching effects on neighboring FAM moieties. Overall, these studies provide insight into practical implications for rapid dsprobe screening and target detection by combining flow cytometry with FRET-based detection.

Low-autofluorescence, transparent composite for multiphoton 3D printing

Multiphoton lithography allows the high resolution, free-form 3D printing of structures such as micro-optical elements and 3D scaffolds for Tissue Engineering. A major obstacle in its application in these fields is material and structure autofluorescence. Existing photoresists promise near zero fluorescence at the expense of poor mechanical properties, and low printing efficiency. Sudan Black B is a molecular quencher used as a dye for biological studies and as means of decreasing the autofluorescence of polymers. In our study, we report the use of Sudan Black B both as a photoinitiator and as a post-fabrication treatment step, using the zirconium silicate SZ2080 for the development of a non-fluorescent composite. We use this material for the 3D printing of micro-optical elements, and meso-scale scaffolds for mesenchymal stem cell cultures. Our results show that the Sudan Black B photosensitive hybrid can be used for the fabrication of high resolution, highly transparent, autofluorescence-free microstructures.

Two-Photon DNAzyme-Gold Nanoparticle Probe for Imaging Intracellular Metal Ions.

RNA-cleaving DNAzymes have been demonstrated as a promising platform for sensing metal ions. However, the poor biological imaging performance of RNA-cleaving DNAzyme-based fluorescent probes has limited their intracellular applications. Compared with traditional one-photon fluorescence imaging, two-photon (TP) fluorescent probes have shown advantages such as increased penetration depth, lower tissue autofluorescence, and reduced photodamage. Herein, for the first time, we developed an RNA-cleaving DNAzyme-based TP imaging probe (TP-8-17ES-AuNP) for Zn2+ detection in living cells by modifying a Zn2+-specific DNAzyme (8-17) with a TP fluorophore (TP-8-17ES) and using gold nanoparticles (AuNPs) for intracellular delivery. The modified TP-8-17ES exhibits good two-photon properties and excellent photostability. For the TP-8-17ES-AuNP, in the absence of Zn2+, the TP fluorophore is quenched by both AuNPs and the molecular quencher. Only in the presence of Zn2+ does the DNAzyme cleave the TP fluorophore-labeled substrate strand, resulting in fluorescence enhancement and TP imaging. Such probe shows remarkable selectivity of Zn2+ over other metal ions existing in the biological environment. Benefiting from the labeled TP fluorophore, the near-infrared (NIR) excited probe has the capability of TP imaging of Zn2+ in living cells and tissue with a deep tissue penetration up to 160 μm. This method can be generally applied to detect other metal ions in biological systems under TP imaging with higher tissue penetration ability and lower phototoxicity.

Reversible Modulation of the Electrostatic Potential of a Colloidal Quantum Dot through the Protonation Equilibrium of Its Ligands.

This Letter describes the reversible modulation of the electrostatic potential at the interface between a colloidal PbS quantum dot (QD) and solvent, through the protonation equilibrium of the QD's histamine-derivatized dihydrolipoic acid (DHLA) ligand shell. The electrostatic potential is sensitively monitored by the yield of photoinduced electron transfer from the QD to a charged electron acceptor, 9,10-anthraquinone-2-sulfonate (AQ). The permeability of the DHLA coating to the AQ progressively increases as the average degree of protonation of the ligand shell increases from 0 to 92%, as quantified by 1H NMR, upon successive additions of p-toluenesulfonic acid; this increase results in a decrease in the photoluminescence (PL) intensity of the QDs by a factor of 6.7. The increase in permeability is attributable to favorable electrostatic interactions between the ligands and AQ. This work suggests the potential of the combination of near-IR-emitting QDs and molecular quenchers as robust local H+ sensors.

A DNAzyme-gold nanoparticle probe for uranyl ion in living cells.

DNAzymes have shown great promise as a general platform for detecting metal ions, as many metal-specific DNAzymes can be obtained using in vitro selection. While DNAzyme-based metal sensors have found many applications in the extracellular environment, no intracellular application of DNAzyme sensors has yet been reported. Here, we demonstrate a novel type of metal ion sensor for intracellular metal ion detection. The probe consists of a 13 nm gold nanoparticle (AuNP) core functionalized with a shell consisting of a uranyl-specific 39E DNAzyme whose enzyme strand contains a thiol at the 3' end for conjugation to the AuNP, and whose substrate strand is modified with a Cy3 fluorophore at the 5' end and a molecular quencher at the 3' end. In the absence of uranyl, the fluorescence of the Cy3 is quenched by both AuNP and the molecular quencher. In the presence of uranyl, the DNAzyme cleaves the fluorophore-labeled substrate strand, resulting in release of the shorter product strand containing the Cy3 and increased fluorescence. We demonstrate that this DNAzyme-AuNP probe can readily enter cells and can serve as a metal ion sensor within a cellular environment, making it the first demonstration of DNAzymes as intracellular metal ion sensors. Such a method can be generally applied to the detection of other metal ions using other DNAzymes selected through in vitro selection.

Molecular Beacon Lighting up on Graphene Oxide

A molecular beacon (MB) is comprised of a fluorophore and a quencher linked by a DNA hairpin. MBs have been widely used for homogeneous DNA detection. In addition to molecular quenchers, many nanomaterials such as graphene oxide (GO) also possess excellent quenching efficiency. Most reported fluorescent sensors relied on DNA probes physisorbed by GO, which may suffer from nonspecific probe displacement and false positive signal. In this work, we report the preparation and characterization of a MB using graphene oxide (GO) as quencher, where an amino and FAM (6-carboxyfluorescein) dual labeled DNA was covalently attached to GO via an amide linkage. A major challenge was to remove noncovalently attached probes due to strong DNA adsorption by GO. While DNA desorption was favored at low salt, high pH, high temperature, and by using organic solvents, the cDNA was required to achieve complete desorption of noncovalently linked DNA probes. The DNA adsorption energy was measured using isothermal titration calorimetry, revealing the heterogeneous nature of GO. The covalent probe has a detection limit of 2.2 nM using a sample volume of 0.05 mL. With a 2 mL sample, the detection limit can reach 150 pM. The covalent probe is highly resistant to nonspecific probe displacement and will find applications in serum and cellular samples where high probe stability is demanded.

Collisional Quenching of Excited Electronic Species and the Parmenter et al. Correlation

The model of collision-induced deactivation of excited species proposed by Parmenter and co-workers has been examined in its application to the collisional quenching of PH(2)(A(2)A(1)). The correlation between the quenching efficiency and the molecular properties of the quenchers is seen to be closely obeyed only by rare gases and two molecules, H(2) and CH(4). The other molecular quenchers, conversely, show only a very loose trend of variation with the quantity epsilon(M-M), well depth of the potential between two molecules of the quencher M, which constitutes the basis for application of the Parmenter et al. model. This apparent limited validity of the Parmenter et al. correlation has been ascribed to the fact that the combining rule applied to the well depth epsilon(PH(2)*-M) of the collision complex PH(2)*-M to express this quantity as (epsilon(PH(2)*-PH(2)*))(1/2)(epsilon(M-M))(1/2) is applicable only to rare gases and not to most of the other quenchers. A procedure to find the characteristics of rare gas-like molecules with quenching efficiencies equal to those of the real quenchers has been proposed. A plot of the quenching data vs (epsilon(Mh-Mh)/k)(1/2) of these hypothetical molecules M(h) with the exception of NO, PH(3), NH(3), and H(2)S shows data points lying on the rare gas line. This indicates that the Parmenter et al. correlation is again verified and that the quenching mechanism of the corresponding real quenchers is the same as that of the rare gases. For NO, PH(3), NH(3), and H(2)S it has been demonstrated that chemical reactions should play an important role in the removal of PH(2)(A(2)A(1)), and the relative parts of physical and chemical quenchings have been evaluated. It has also been possible to determine the otherwise inaccesible quantity epsilon(PH(2)*-M), i.e., the well depth of the collision complexes, present in the original equation of the Parmenter et al. model. By plotting the quenching data vs epsilon(PH(2)*-M)/k, it can be seen again that the Parmenter et al. model is followed by all the quenchers treated, except the above-mentioned molecules.

Water-soluble phosphate-functionalized polyfluorene as fluorescence biosensors toward cytochrome c

An anionic water-soluble polyfluorene derivative, poly(9,9-bis(6′-phosphatehexyl)fluorene-alt-1,4-phenylene) sodium salt (PFHPNa), was synthesized by Suzuki coupling reaction in DMF/water. Polymer PFHPNa was well soluble in water with a strong blue fluorescence emission. Effect of the side chain length on fluorescence sensory properties was studied by comparing quenching efficiencies toward different quenchers of PFHPNa with a reported polymer poly(9,9-bis(3′-phosphatepropyl)fluorene-alt-1,4-phenylene) sodium salt (PFPPNa), which have different side chains in length. For small molecular quenchers (methylviologen, MV2+) and meso-5,10,15,20-tetrakis-(N-methyl-4-pyridyl)porphine (TMPyP4), polymer PFHPNa had lower sensitivity due to the much longer side chain length. The positively charged metalloprotein cytochrome c could quench fluorescence of conjugated polymers via energy transfer and electron transfer. Moreover, polymer PFHPNa showed higher fluorescence quenching toward large biomolecules than PFPPNa. The corresponding Stern-Volmer (K sv) value of polymer PFHPNa was determined to be 2.1×108 M−1 for cytochrome c. It could be used as a sensitive and selective fluorescence sensor for protein cytochrome c.

Water-Soluble Multifunctional Cross-Conjugated Poly(p-phenylenes) as Stimuli-Responsive Materials: Design, Synthesis, and Characterization

The design, synthesis, and characterization of a series of thermally stable water-soluble polymers with cross-conjugation and multiple functional groups are reported. All copolymers were characterized by means of FT-IR, GPC, 1H and 13C NMR, UV−vis, and fluorescence spectroscopy. Photophysical properties showed collective responses from their respective conjugated segments. Titration studies showed that such polymers have good sensitivity for both cationic and anionic quenchers in fluorescence decay, with an average Stern−Volmer constant of 105 M-1. Besides fluorescence quenching, significant shifts in absorption and emission maxima upon addition of small molecules were also observed. High concentration of small molecular quenchers induced aggregation of the polymers. All polymers were able to form highly fluorescent, flexible, free-standing thin films via evaporation of the solvent. Hence, by attaching cationic and anionic groups on a cross-conjugated water-soluble structure, a multifunctional CP system was developed for potential applications.

Adsorption and Photophysics of Fullerene C60 at Liquid−Zeolite Particle Interfaces: Unusually High Affinity for Hydrophobic, Ultrastabilized Zeolite Y

Adsorption of fullerene C60 from solution to the external surface of zeolite particles has been investigated. The most intriguing result of this study was the nature of C60 adsorption to ultrastablized zeolite Y (or USY). Two commercial samples of USY were tested: CBV780 (Y780) and CBV901 (Y901). Y901 was shown in previous reports to be more hydrophobic than Y780. Higher affinity of C60 for Y901 was found relative to Y780 in a variety of hydrocarbon solvents, including toluene and cyclohexane. In these same solvents, weak or no affinity for Y901 of typical arenes such as naphthalene or pyrene was observed. In toluene, adsorption isotherms for C60 gave dissociation constants (and values of saturation binding) = 0.5 microM (5.8 micromol g(-1)) and 8 microM (1.4 micromol g(-1)) for Y901 and Y780, respectively. C60 was estimated to cover nearly one-half of the estimated external surface area of Y901 particles at saturation. Significant adsorption of C60 to the ionic zeolites NaX, NaY, and KL was observed in cyclohexane but not in toluene, consistent with the pi-cation effect as a driving force for adsorption to these materials. The main driving force for C60 adsorption to Y901 is postulated to involve the interaction of C60 with lone pair electrons of framework oxygen atoms of the 12-ring entry aperture to the supercage. In the 12-ring site, C60 is located in half-supercage bowls on the exterior particle surface. The adsorptive interaction on Y901 relies on the spherical shape of C60 and the hydrophobicity of the zeolite surface. On ionic zeolites, the presence of specific adsorption sites such as exchangeable cations and hydroxyl groups hinder the special positioning necessary for C60 interaction with the 12-ring site. The ground-state and triplet-state absorption spectrum of adsorbed C60 was solution-like on all zeolites. Quenching of the C60 triplet state was examined by using transient absorption spectroscopy. Rate constants for quenching by rubrene, ferrocene, and O2 at the Y901-toluene interface were 18, 9, and 3 times lower, respectively, relative to rate constants in solution. These differences point out that the approach of molecular quenchers to C60 at the interface is more hindered for larger molecules, an expected result for C60 located in half-supercage bowls. The high affinity of fullerenes for hydrophobic zeolite Y provides a strategy for organizing fullerenes at interfaces and for studies of fullerene photochemistry.

Collisional deexcitation of the excited rare gas atoms in resonant states: The Watanabe–Katsuura theory revisited

The cross sections for the collisional deexcitation of neon atoms in the lowest excited P11 state by Ar, Kr, Xe, N2, O2, CO, NO, and CH4, and in the lowest excited P13 state by O2 and CH4 have been measured at a mean collisional energy corresponding to room temperature. Data are also included for collisions of argon atoms in the lowest excited P11 and P13 states by C2H4, cyclo-C3H6, and C3H8, and collisions of krypton atoms in the lowest excited P11 and P13 states by C2H4 and cyclo-C3H6. The measured cross sections, together with those obtained in our previous studies, are compared with the cross sections calculated using the Watanabe–Katsuura theory. An extension of the Watanabe–Katsuura theory to the deexcitation of excited rare gas atoms in collisions with molecular quenchers, not atoms, is examined.

Collision induced deactivation of PH2(Ã 2A1;v2′=1,0) and PH2(X̃ 2B1;v2″=1) by diatomic molecules

Deactivation of the bending mode v2′=1 and v2′=0 vibrational levels of PH2(Ã 2A1), and of the v2″=1 level of ground state PH2(X̃ 2B1) due to collisions with the diatomic molecules H2, N2, CO, and NO has been investigated. The Parmenter and co-worker’s, the Thayer and Yardley’s, and the collision complex theories have been used to rationalize the PH2(Ã 2A1) quenching data. Explanations for the deviations of the quenching data due to these molecular quenchers from the variation trend found, in a previous work, for the rare gas quenchers have been proposed. For the vibrational relaxation of PH2(X̃ 2B1;0,1,0), the data interpretation is based essentially on the theories by Schwartz, Slawsky, and Herzfeld–Tanczos, and Moore.

State-specific collisional energy transfer in electronically excited SiF radicals: dramatic contrasts with SiCl

The collisional removal of state-selected SiF (C 2Δ, υ′ = 0 or 1) has been studied by time- and wavelength-resolved laser-induced fluorescence spectroscopy. A series of non-polar quenchers (Ar, He, H 2, N 2, CO 2 and CH 4) was examined. Rate constants are generally large for the molecular quenchers, but are negligible for the noble gases. A collisionless radiative lifetime of 94±2 ns (2σ statistical uncertainty) is also reported for the SiF C 2Δ state, independent of vibrational level. A fraction of the removed C 2Δ excited valence state population, varying from 0 to 20% with quencher, is deposited in the nearby B 2Σ + Rydberg state. The resultant B 2Σ + state vibrational populations are largely quencher independent and correlate well with the Franck-Condon overlap between the initial and product vibronic states, despite the substantial energy gaps involved. These results contrast dramatically with those for the nominally isoelectronic states in SiCl: possible explanations for the distinctions are discussed.

State-to-state studies of the collisional quenching of electronically excited Cd(1P1) atoms

A comprehensive study of the quenching of electronically excited Cd (5s5p 1P1) by a variety of simple molecules has been conducted using pulsed laser techniques. Except for He, Ar, and C2F6, deactivation occurs at essentially every encounter with all quenchers studied. The following process occurs with high efficiency with most of the quenching molecules studied, in striking violation of the Wigner spin rule and in spite of other available chemical or energy-transfer exit channels in many cases: Branching ratios for total Cd(5s5p3PJ) production were determined using an indirect method calibrated by earlier absolute measurements. Initial distributions of individual Cd(3P2,1,0) quantum states in process (1), determined by a pump-and-probe laser-induced fluorescence technique, show wide variations for different types of molecular quenchers. A qualitative model of bond-specific interactions of Cd(1P1) with quenching molecules has been developed which successfully rationalizes all the experimental results. For several of the molecules, and electronically statistical (5:3:1) distribution of Cd(3P2,1,0) is observed and attributed to a preponderance of bond sites at which there is a net attractive interaction of Cd(1P1), caused either by intersection of ionic surfaces (N2,CO) or by chemical bonding (alkyl C–H bonds), so that crossings occur with equal probability to all repulsive states correlating with Cd(3P2,1,0). Cases for which the Cd(3P2,1,0) distribution is skewed away from statistical towards Cd(3P2) [Ar, CH4, C2H6, C(CH3)4, C3H8] result from a less attractive potential with Cd(1P1) (due to stronger C–H bonds and/or weaker dispersion forces) and preferential crossings with the most repulsive ’’S’’-like states, which correlate with Cd(3P2,1,0). Cases for which the Cd(3P2,1,0) distribution is skewed away from statistical towards Cd(3P1) and Cd(3P0) (NO, C3F6, C2H2, C2H4, butadiene, propylene) are postulated to result from formation of long-lived complexes and the resultant formation of products which approach truly statistical distributions of vibrational, rotational, translational, and electronic energy. For the higher alkenes (isobutylene, 2-butene), Cd(1P1) interaction with the alkly C–H bonds completely dominates over the double bond interaction and electronically statistical Cd(3P2,1,0) distributions are observed.