Fluorescence Quenching Research Articles

Development of a colorimetric and turn-on fluorescent probe with large Stokes shift for H2S detection and its multiple applications in environmental, food analysis and biological imaging

A new colorimetric and turn-on fluorescent probe NPC-H2S with a large Stokes shift (227 nm) was designed and synthesized for the detection of hydrogen sulfide (H2S) in environmental, food analysis and biological imaging. NPC-H2S was strategically constructed by employing a new corrole derivate (NPC–OH) as the fluorophore and a benzenesulfonyl group (DNBS) as the identification moiety and fluorescence quencher. NPC-H2S displays high selectivity and sensitivity for H2S over various analytes with fluorescent “off-on” ultra-fast recognition (10 s), a low limit of detection (61 nM), and a naked-eye colorimetric change from green to yellow. Importantly, NPC-H2S has been successfully applied for the detection of H2S in the actual water samples and monitoring the spoilage of food samples such as chicken, beef, pork, fish, cracked egg and full egg, as well as for fluorescent bioimaging of H2S in living cells and zebrafish.

Quantum-Classical Protocol for Efficient Characterization of Absorption Lineshape and Fluorescence Quenching upon Aggregation: The Case of Zinc Phthalocyanine Dyes.

A quantum-classical protocol that incorporates Jahn-Teller vibronic coupling effects and cluster analysis of molecular dynamics simulations is reported, providing a tool for simulations of absorption spectra and ultrafast nonadiabatic dynamics in large molecular photosystems undergoing aggregation in solution. Employing zinc phthalocyanine dyes as target systems, we demonstrated that the proposed protocol provided fundamental information on vibronic, electronic couplings and thermal dynamical effects that mostly contribute to the absorption spectra lineshape and the fluorescence quenching processes upon dye aggregation. Decomposing the various effects arising upon dimer formation, the structure-property relations associated with their optical responses have been deciphered at atomistic resolution.

Violet phosphorene nanosheets coupled with CRISPR/Cas12a in a biosensor with a low background signal for onsite detection of tigecycline-resistant hypervirulent Klebsiella pneumoniae

CRISPR/Cas12a-based detection has been widely used for DNA targets, but always suffers from a relatively high background fluorescent signal, which potentially gives false positive results, and limits the accuracy and specificity of detection. To address this problem, violet phosphorene nanosheets (VPNSs) with excellent DNA adsorption and light absorption capacities were synthesized by liquid exfoliation from bulk violet phosphorus. The introduction of VPNSs into CRISPR/Cas12a-based detection decreased the relatively high background fluorescent signal by 32.7 %, and resulted in a background with the relative fluorescence unit of 2590.5 that was no longer visible via a visual readout, demonstrating that VPNSs are efficient fluorescence quenchers. By combining the detection with a smartphone-based fluorescence imaging system (FIS), a biosensor was developed for simultaneous detection of virulence genes iucA, iroB, peg-344, rmpA and rmpA2, and resistance gene tet(A) carried by tigecycline-resistant hypervirulent Klebsiella pneumoniae (HvKP). Upon optimization, the VPNSs-CRISPR/Cas12a-FIS biosensor detected 1 cfu of tigecycline-resistant HvKP in 60 min with a low background signal. The whole process was performed with portable devices including a block heater and an FIS, and the signal could be observed with either a smartphone or the naked eye. The biosensor provides new insights for exploration of low background, multiple target, and onsite analytical methods based on CRISPR/Cas12a.

FRET with MoS2 nanosheets integrated CRISPR/Cas12a sensors for robust and visual food-borne parasites detection

Recently, CRISPR/Cas associated biosensors have been shown to have great potential in sensing applications due to their high sensitivity and high base resolution. However, the signal reporter system containing two organic fluorescent dye pair is limited by high cost and less stability. In contrast, functional nanomaterials exhibit robust stability, excellent optical properties and low preparation cost, making them suitable reporters. In this study, a MoS2 nanosheets (NSs) improved CRISPR/Cas12a-based biosensing platform was constructed for the first time to detect food-borne parasites. MoS2 NSs were used as fluorescence quenchers and single-stranded DNA (ssDNA) discriminated carrier to construct CRISPR/Cas signal reporter system. The combination of recombinase polymerase amplification with MoS2 NSs modulated CRISPR/Cas12a helped achieve attomolar sensitivity for nucleic acid detection within 35 min. Moreover, the results were obtained using a portable apparatus, enabling visual detection at the point of care. The practical applicability of this biosensing platform was successfully achieved through the detection of anisakis in real samples. This study provides novel insights into exploring the feasibility of two-dimensional nanomaterials based reporter in the CRISPR/Cas12a system, as well as offers a reliable tool for on-site monitoring of parasites.

Target-induced multipath-to-one-substrate approach for high-efficient bioanalysis of microRNA

Considering the significant potential of microRNA (miRNA) as an efficient biomarker and great challenge of accurate analysis of lowly abundant miRNA, herein, we proposed a target-induced multipath-to-one-substrate strategy to monitor miRNA in vivo and in vitro accurately with high-efficient performances. In presence of target miRNA, it could directly generate the catalytic hairpin assembly (CHA) amplification cycle based on hybridizing with hairpin 1 (H1) and H2 respectively to structure the H1–H2 duplex, then the H1–H2 duplex could activate the cleavage ability of CRISPR/Cas12a to cleavage H1 which represent miRNA indirectly consume H1, which achieve co-consumption of the same substrate H1 by multiple pathways. And thus, the quenched fluorescent signal on H1 could be recovered due to the enlarger distance between fluorescent probe and quencher by the formation of H1–H2 duplex or cleavage of H1, all of which were related directly with target miRNA or indirectly with H1–H2 duplex activated cleavage ability of CRISPR/Cas12a, generating ultrahigh sensitive analytical ability and high-efficient analytical performances, such as more simple, fast, efficient and so on, especially a linear correlation from 100 pM to 100 nM with a detection limit of 78 pM, opening a new door to monitor expression level of biomolecules for early diagnosis and prognosis evaluation of various diseases.

3-Ferrocenyl-estra-1,3,5 (10)-triene-17-one: Synthesis, Crystal Structure, Hirshfeld Surface Analysis, DFT Studies, and Its Binding to Human Serum Albumin Studied through Fluorescence Quenching and In Silico Docking Studies.

3-ferrocenyl-estra-1,3,5 (10)-triene-17-one (2), [Fe(C5H5)(C24H25O3)], crystallizes in the monoclinic space group C2. The cyclopentadienyl (Cp) rings adopt a nearly eclipsed conformation, and the Cp plane is tilted by 87.66° with respect to the substituted phenyl plane. An average Fe-C(Cp) bond length of 2.040(13) Å was determined, similar to the one reported for ferrocene. Hirshfeld surfaces and two-dimensional fingerprint plots were generated to analyze weak intermolecular C-H···π and C-H···O interactions. Density functional theory studies revealed a 1.15 kcal/mol rotational barrier for the C3-O1 single bound. Fluorescence quenching studies and in silico docking studies suggest that human serum albumin forms a complex with 2 via a static mechanism dominated by van der Waals interactions and hydrogen bonding interactions.

Detection of TNT Vapors by Fluorescence Quenching and Self-Recovery Using Highly Fluorescent Conjugated Silole Polymers

Detection of TNT Vapors by Fluorescence Quenching and Self-Recovery Using Highly Fluorescent Conjugated Silole Polymers

STR genotyping by real-time PCR using QueSTR probes

QueSTR probes provide a feasible alternative for implementing forensic Short Tandem Repeat (STR) genotyping on lab-on-a-chip (LoC) devices. Unlike capillary electrophoresis (CE), which poses challenges for LoC implementation, QueSTR probes have been demonstrated to accurately genotype STRs in a hybridization curve assay after PCR amplification. In this study, we modified the QueSTR probe assay for use as a hydrolysis probe assay during qPCR. An asymmetric real-time PCR was performed with QueSTR probes and RNase H2 in the master mix, during which the fluorescence was recorded. Designed to hybridize with specific STR alleles, QueSTR probes contain a fluorescent dye and quencher molecule, enabling detection of probe hybridization through RNase H2-mediated cleavage of the RNA:DNA duplex, which releases the quencher. Matching probes yielded lower threshold cycle values and steeper incline of fluorescence curves compared to non-matching probes, indicating accurate genotype. The QueSTR qPCR assay was used to successfully genotype four CODIS core loci (D16S539, D7S820, TPOX, and TH01) in 12 samples, with one exception. The integration of amplification and detection in a single reaction supports the use of QueSTR probes for miniaturizing STR genotyping, thereby complementing CE-based analyses in centralized labs.

Improved Characteristics of Fluorene‐Type Polymer Light‐Emitting Devices Based on Multilayer Formation from Polymers and Solution‐Processable Wide‐Bandgap Inorganic Copper(I) Thiocyanate with p‐Type Conduction and High Refractive Index

The functional solution‐processable multilayer formation is a significant factor in the improved characteristics of polymer light‐emitting devices. The improved characteristics of direct current and alternating current (AC)‐driven fluorene‐type polymer light‐emitting devices based on multilayer structures utilizing polymers and solution‐processable inorganic wide‐bandgap semiconductor, copper(I) thiocyanate, CuSCN, which exhibits p‐type conduction and high refractive index, are investigated. For the polymer light‐emitting diodes based on poly(9,9‐dioctylfluorene‐co‐benzothiadiazole), F8BT with CuSCN, the plateau of current efficiency indicates the carrier injection and transport to be balanced at lower current density and the balance to be maintained through the wide current range. CuSCN acts as the hole transport and electron‐blocking layer, although CuSCN acts as a fluorescence quencher for F8BT. The introduction of inorganic/organic hybrid dielectric mirrors, which consists of the alternating layers of inorganic CuSCN and insulating poly(vinylidene fluoride‐trifluoroethylene), P(VDF‐TrFE), into the AC voltage‐driven polymer electroluminescent device (ACEL) structure, is an effective way to achieve both the spectral narrowing and color tunability. Using high‐refractive‐index CuSCN, the color‐tunable emission for ACEL with distributed Bragg reflector mirrors and the electroluminescent (EL) emission from a weak microcavity effect are achieved.

A Ratiometric Fluorescent Probe Based on RhB Functionalized Tb-MOFs for the Continuous Visual Detection of Fe3+ and AA.

In this study, a red-green dual-emitting fluorescent composite (RhB@MOFs) was constructed by introducing the red-emitting organic fluorescent dye rhodamine B (RhB) into metal-organic frameworks (Tb-MOFs). The sample can be used as a ratiometric fluorescent probe, which not only avoids errors caused by instrument and environmental instability but also has multiple applications in detection. The results indicated that the RhB@MOFs exhibited a turned-off response toward Fe3+ and a turned-on response for the continuous detection of ascorbic acid (AA). This ratiometric fluorescent probe possessed high sensitivity and excellent selectivity in the continuous determination of Fe3+ and AA. It is worth mentioning that remarkable fluorescence change could be clearly observed by the naked eye under a UV lamp, which is more convenient in applications. In addition, the mechanisms of Fe3+- and AA-induced fluorescence quench and recovery are discussed in detail. This ratiometric probe displayed outstanding recognition of heavy metal ions and biomolecules, providing potential applications for water quality monitoring and biomolecule determination.

Determination and Quantification of Cetirizine Dihydrochloride in Pharmaceutical Formulations and Biological Fluids Through Fluorescence Quenching of Eosin Y: Application to Content Uniformity Test

Determination and Quantification of Cetirizine Dihydrochloride in Pharmaceutical Formulations and Biological Fluids Through Fluorescence Quenching of Eosin Y: Application to Content Uniformity Test

Dual-signal and one-step monitoring of Staphylococcus aureus in milk using hybridization chain reaction based fluorescent sensor

Food-borne pathogens in dairy products that was contaminated from raw ingredients or improper food handling can cause a major threaten to human health. Here, to construct the pathogens detection, a dual-signal readout fluorescent switching sensor was designed for one-step determination of Staphylococcus aureus (S. aureus), which was a marker of food contamination. Graphene oxide (GO) was used as a fluorescence quencher, while fluorophore-labeled hairpin DNA was used as a donor, resulting in fluorescence resonance energy transfer (FRET) from the fluorophore to GO (signal off). Enzyme-free hybridization chain reaction could generate remarkable signal amplification, which avoided the nonspecific desorption caused by any enzymatic proteins in GO surface. With the strong binding ability of aptamer to S. aureus, a long bifluorescent molecules-labeled double-stranded DNA product was formed, bringing in dual-signal readout responses (signal on). Consequently, a reliable, sensitive and selective sensor was obtained for one-step quantification of S. aureus concentration from 10 to 108 CFU/mL with a detection limit of 1 CFU/mL. Furthermore, satisfactory stability, reproducibility, specificity and good recovery efficiency in milk samples revealed that the proposed sensor could be served as a prospective tool for food safety analysis.

Synthesis, Crystal Structure, DFT and Fluorescence Quenching Study of Novel syringe aldehyde-derived hydrazinyl-imidazole Based Schiff base Chemosensor.

In this study, we report a new syringe aldehyde-derived hydrazinyl-imidazole basedfluorescent sensor (L) for sensitive detection of different inorganic quenchers (halide ions, bicarbonate ion, sulphide ion and transition metal ions). The chromophore (L) was obtained in good yield by the 1:1 condensation reaction of 2-hydrazino-4,5-dihydroimidazole hydrobromide and 4-hydroxy-3,5-dimethoxy benzaldehyde.L exhibited strong fluorescence in the visible region (around 380nm) and its interaction with different quenchers was studied in details via fluorescence technique. For the halide ions series, its sensitivity is higher for NaF (Climit = 4 × 10- 4M) than for NaCl while the fluorescence quenching occurred mainly through a dynamic process. Similar considerations were observed for HCO3- and S2- quencher too, when static and dynamic quenching take place simultaneously. Regarding transition metal ions, at a fixed ion concentration (4 × 10- 6M), best performance was achieved for Cu2+ and Fe2+ (fluorescence intensity was reduced by 79% and 84.9% respectively), while for other metal ions, the sensor performance was evaluated and found to be very less (< 40%). Thus, minimum detection limits (10- 6 - 10- 5M range) recommended the use of such derivatives as highly sensitive sensors capable to monitor delicate changes in varied environments.

Measuring the Surface Area Concentration and Specific Surface Area of Mass-Produced Graphene Nanoflakes via Fluorescence Quenching

A primitive cubic lattice composed of 1,000 atoms has 488 surface sites. By definition, every atom in a strictly two-dimensional single-layer lattice composes its surface. These surface atoms are the ones that undergo chemical interactions with the surrounding medium, thereby defining the functionalities of the nanostructure. As such, one of the most important morphological properties of nano-objects is the extremely large specific surface area that enhances their levels of reactivity. Here, we introduce an optical spectroscopy method to measure the surface area concentration, ρA, of mass-produced graphene nanoflakes in liquid dispersions. The information is accessed from the quenching of the fluorescence signal from the dye molecules dispersed in the medium. We found that the quantum efficiency of the fluorescence signal decays exponentially with the concentration of graphene’s surface area, the decay rate being independent of the degree of exfoliation. If the mass concentration ρ is known by other means, the specific surface area can be extracted from the ratio ρA/ρ. The measurements can be performed directly in liquid suspensions of nanoflakes, being highly applicable to the quality control of mass-produced two-dimensional nanomaterials, especially by means of mechanically assisted liquid-phase exfoliation.

Disposable Device for Bacterial Vaginosis Detection.

Due to the increasing demand for clinical testing of infectious diseases at the point-of-care, the global market claims alternatives for rapid diagnosis tools such as disposable biosensors, avoiding the need for specialized laboratories and skilled personnel. Bacterial vaginosis (BV) is an infectious disease that commonly affects reproductive-age women and predisposes the infection of sexually transmitted diseases. Especially in asymptomatic cases, BV can lead to pelvic inflammatory conditions, postpartum endometritis, and preterm labor. Conventionally, BV diagnosis involves the microscopic analysis of vaginal swab samples; it thus requires highly trained personnel. In response, we report a novel microfluidic paper-based analytical device for BV diagnosis. Sialidase, a biomarker overexpressed in BV, was detected by exploiting an immunosensing mechanism previously discovered by our team. This technology employs a graphene oxide-coated surface as a quencher of fluorescence; the fluorescence of the immunoprobes that do not experiment immunoreactions (antibody-antigen) are deactivated by graphene oxide via non-radiative energy transfer, whereas those immunoprobes undergoing immunoreactions preserve their photoluminescence due to the distance and the low affinity between the immunocomplex and the graphene oxide-coated surface. Our paper-based test was typically carried out within 20 min, and the sample volume was 6 μL. Besides, it was tested with 14 vaginal swabs specimens to discriminate clinical samples of women with normal microbiota from those with BV. Our disposable device represents a new tool to prevent the consequences of BV.

Vermiculite-derived cationic molecular-intercalated fluorescent MgAl-LDHs for assembling ultrathin solid-state light-emitting films

Solid-state light-emitting devices are of special interest in optoelectronic applications, owing to inherent advantages in terms of low cost, high efficiency and desirable durability. However, the accumulation of solid fluorescent chromophores causes serious energy transfer, resulting in fluorescence quenching. Herein, exfoliated vermiculite (VMT) was structurally disassembled into Mg-O octahedral and Si-O tetrahedral layers to produce hydrotalcite (LDHs) and SiO2, respectively, realizing efficient utilization of each component and VMT surface. The fluorescent intercalated LDHs was intercalated with organic fluorescent dyes, including rhodamine 6G (R6G), pyronine G (PG) and sodium fluorescein (Fl-Na), to prepare high-performance fluorescent LDHs, which was further assembled with VMT nanosheets to form ultrathin solid-state light-emitting films. Based on the electrostatic self-assembly strategy, the localization of chromophores and light-emitting intensity of solid films can be precisely controlled by adjusting ordered multilayers. In fact, the exfoliated VMT could cause the quenching of organic fluorescent dyes in liquid, therefore VMT can be used as fluorescent quencher, which could be regenerated by heating. The designed assembly strategy avoids the fluorescence quenching of VMT. Without the addition of any surfactant, both anionic and cationic organic fluorescent dye can be intercalated into hydrotalcite, overcoming the disadvantage of LDHs that can only intercalate anionic molecules. In particular, the VMT encapsulation overcomes the quenching problem of fluorescent dye and improves the stability of the light-emitting films. This work provides a potential strategy for assembling solid-state light-emitting films and opens new applications in various optoelectronics, such as surface-emitting light source and full-color flat-panel displays.

Binding of Apo and Glycated Human Serum Albumins to an Albumin-Selective Aptamer-Bound Graphene Quantum Dot Complex.

Diabetes mellitus is a chronic metabolic disease involving continued elevated blood glucose levels. It is a leading cause of mortality and reduced life expectancy. Glycated human serum albumin (GHSA) has been reported to be a potential diabetes biomarker. A nanomaterial-based aptasensor is one of the effective techniques to detect GHSA. Graphene quantum dots (GQDs) have been widely used in aptasensors as an aptamer fluorescence quencher due to their high biocompatibility and sensitivity. GHSA-selective fluorescent aptamers are first quenched upon binding to GQDs. The presence of albumin targets results in the release of aptamers to albumin and consequently fluorescence recovery. To date, the molecular details on how GQDs interact with GHSA-selective aptamers and albumin remain limited, especially the interactions of an aptamer-bound GQD (GQDA) with an albumin. Thus, in this work, molecular dynamics simulations were used to reveal the binding mechanism of human serum albumin (HSA) and GHSA to GQDA. The results show the rapid and spontaneous assembly of albumin and GQDA. Multiple sites of albumins can accommodate both aptamers and GQDs. This suggests that the saturation of aptamers on GQDs is required for accurate albumin detection. Guanine and thymine are keys for albumin-aptamer clustering. GHSA gets denatured more than HSA. The presence of bound GQDA on GHSA widens the entrance of drug site I, resulting in the release of open-chain glucose. The insight obtained here will serve as a base for accurate GQD-based aptasensor design and development.

A Study of the Electronic Absorption and Emission Spectra of DBDMA Dye: Solvent Effect, Energy Transfer, and Fluorescence Quenching

This study is aimed to shed light on the electronic absorption and emission spectra of DBDMA (2-(1-(difluoroboraneyl)-1,2-dihydroquinolin-2-yl)-2-(1-methylquinoxalin-2-ylidene) acetonitrile) in different solvents. Both types of spectra were obtained theoretically and produced experimentally in different solvents. The photostability of dye was tested, and its energy transfer behavior in the presence of oxygen and hydrated copper sulfate quenchers was investigated. We also gave a qualitative estimation of the effect of acidic media on the absorption and emission spectra. There is good compatibility between the calculated and measured values of many photophysical parameters. DBDMA has a low chemical quantum yield in solvents of different polarities, and the fluorescence quantum yield is high enough, which confirms, together with the low values of the excited state lifetime, its efficiency as laser emitting dye in the range of wavelength emission maxima. The rigidity of the DBDMA molecule is the main reason for the photochemical stability and the absence of a considerable shift as a result of the change in the polarity of the solvents. Geometries of ground and excited states were optimized using the density functional theory (DFT) and the time-dependent density functional theory (TD-DFT), respectively. Upon using the TD-DFT method, the UV-Vis absorption and emission spectra of the DBDMA molecule in different solvents were illustrated. A slight change is observed in the position of the maximum emission and absorption wavelength with the change of the solvent due to the rigidity of the compound. There was no apparent effect of quenching by oxygen. Besides, no intersystem crossing (ISC) was observed for the excited state of the DBDMA as a result of aeration of a solution with O2 for 20 min, which was an explanation of the stability of peak emission intensity of dye after exposure to oxygen gas. The energy transfer rate constant has been calculated as well.

Rapid detection of intact SARS-CoV-2 using designer DNA Nets and a pocket-size smartphone-linked fluorimeter.

Rapid, sensitive, and inexpensive point-of-care diagnosis is vital to controlling highly infectious diseases, including COVID-19. Here, we report the design and characterization of a compact fluorimeter called a "Virus Pod" (V-Pod) that enables sensitive self-testing of SARS-CoV-2 viral load in saliva. The rechargeable battery-operated device reads the fluorescence generated by Designer DNA Nanostructures (DDN) when they specifically interact with intact SARS-CoV-2 virions. DDNs are net-shaped self-assembling nucleic acid constructs that provide an array of highly specific aptamer-fluorescent quencher duplexes located at precise positions that match the pattern of spike proteins. The room-temperature assay is performed by mixing the test sample with DNA Net sensor in a conventional PCR tube and placing the tube into the V-Pod. Fluorescent signals are generated when multivalent aptamer-spike binding releases fluorescent quenchers, resulting in rapid (5-min) generation of dose-dependent output. The V-Pod instrument performs laser excitation, fluorescence intensity quantitation, and secure transmission of data to an App via Bluetooth™. We show that the V-Pod and DNA Net assay achieves clinically relevant detection limits of 3.92×103 viral-genome-copies/mL for pseudo-typed wild-type SARS-CoV-2 and 1.84×104, 9.69×104, 6.99×104 viral-genome-copies/mL for pathogenic Delta, Omicron, and D614G variants, representing sensitivity similar to laboratory-based PCR. The pocket-sized instrument (∼$294), inexpensive reagent-cost/test ($1.26), single-step, rapid sample-to-answer, and quantitative output represent a capability that is compatible with the needs of frequent self-testing in a consumer-friendly format that can link with medical service systems such as healthcare providers, contact tracing, and infectious disease reporting.

Persistent Fluorescence Switching of a Probe Using a Photochromic Quencher with High Photostability Assisted by Protein-Surface Modification.

Photoswitchable fluorescent molecules (PSFMs) are widely applicable in the life sciences for super-resolution imaging. Owing to the large and hydrophobic molecular structures of PSFMs that may aggregate in a biological medium, the development of synthetic PSFMs with persistent reversible photoswitching is challenging. Here, we established a protein-surface-assisted photoswitching strategy that allows for persistent reversible fluorescence photoswitching of a PSFM in an aqueous solution. As a first step, we applied the photochromic chromophore furylfulgimide (FF) as a photoswitchable fluorescence quencher and developed a Förster resonance energy transfer-based PSFM, named FF-TMR. Most importantly, the protein-surface modification strategy allows FF-TMR to exhibit persistent reversible photoswitching performance in an aqueous solution. In fixed cells, the fluorescence intensity of FF-TMR bound to antitubulin antibody was repetitively modulated. The protein-surface-assisted photoswitching strategy will be a useful platform to broaden the utility of functionalized synthetic chromophores enabling persistent fluorescence switching that inherits their high resistance to light irradiation.