Significant Fluorescence Enhancement Research Articles

Synthesis and experimental investigations of a photoactive naphthopyran for sensing nanomolar concentration of ammonia

Selective detection and identification of toxic analytes such as trace gases hold significant relevance in supramolecular host–guest functional systems. Identification becomes easier when the detection tool is prone to fluorescence enhancement instead of quenching. A judiciously designed substituted naphthopyran derivative was synthesized and its interactions with several amine guests have been investigated. The probe displayed enhancement in fluorescence signal on interaction with ammonia. Based on fluorescence spectroscopy a limit of detection value of 56 nM was witnessed for ammonia. Molecular level interaction analysis performed using 1H NMR spectroscopy indicated ring opening of naphthopyran derivative induced by ammonia. The designed naphthopyran probe might be useful as a tool for portable NH3 sensing.

Development of time-resolved fluoroimmunoassay with enhanced fluorescence reaction for the quantitation of atezolizumab in plasma.

Atezolizumab (ATZ) is a human monoclonal antibody, which has been granted multiple approvals from the US Food and Drug Administration (FDA) for the immunotherapy of different types of cancer. This study describes the prototype of a time-resolved fluoroimmunoassay (TRFIA) for the quantitation of ATZ in plasma. The assay involved the non-competitive binding of ATZ to its specific antigen [programmed death-ligand 1 (PD-L1) protein]. The immune complex formed on the inner surface of the assay plate wells was quantified by anti-human secondary antibody labeled with a chelate of europium-ethylenediaminetetraacetic acid. The enhanced fluorescence signal was generated by an enhanced fluorescence solution composed of thenoyltrifluoroacetone, trioctylphosphine oxide, and Triton X-100. The conditions of the TRFIA were refined, and its optimum procedures were established. The assay was validated in accordance with the immunoassay validation guidelines, and all the validation parameters were acceptable. The working range of the assay was 20-1000 pg mL-1, and its limit of quantitation was 20 pg mL-1. The assay was applied to the quantitation of ATZ in plasma samples with satisfactory accuracy and precision. The proposed TRFIA has significant benefits over the existing methodologies for the quantitation of ATZ in clinical settings.

Copper(II) Driven Fluorescence switch-on Detection of Ovalbumin and GSH Using a Pyridoxal 5'-phosphate Derived Tetradentate Schiff Base and its Applications.

The facile detection of glutathione (GSH) and ovalbumin (OVA) is of great importance in biological research. Herein, a tetradentate Schiff base N, N'-bis(pyridoxal-5-phosphate)-o-phenylenediamine (L) obtained by condensing two moles of pyridoxal 5'-phosphate (PLP) with one mole of 1,2-phenylenediamine was employed for the fluorescence switch-on detection of GSH and OVA. When excited at 389nm, receptor L showed a weak emission at 454nm in an aqueous medium. The addition of GSH to the solution of L caused a significant fluorescence enhancement at 454nm. Amino acids (leucine, glycine, serine, tryptophan, homocysteine, alanine, methionine, arginine and proline) and albumins (bovine serum albumin and OVA) failed to alter the fluorescence profile of L. Receptor L can be applied to detect GSH down to 1.16 µM. However, the fluorescence emission of L was quenched upon the formation of the L-Cu2+ complex. The addition of GSH and OVA to the in-situ formed L-Cu2+ complex restored not only the fluorescence emission of L but also a noticeable fluorescence enhancement observed at 454nm. The decomplexation of L-Cu2+, along with the interaction of L with GSH and OVA is expected to suppress the conformational flexibility of L that enhanced the fluorescent intensity at 454nm. Using L-Cu2+ complex, the concentration of OVA and GSH can be detected down to 0.31 µM and 0.20 µM, respectively. Molecular docking and dynamics simulation were performed to analyze the binding mode, conformational flexibility and dynamic stability of the L-Cu2+-OVA complex. Finally, the analytical novelty of L-Cu2+ was examined by detecting GSH/OVA in real biological samples, such as human blood serum, urine, and egg white.

Activatable near-infrared fluorescence and chemical exchange saturation transfer MRI multimodal imaging probe for tumor detection in vitro and in vivo

Multimodal imaging has emerged as a powerful tool in biomedical research and clinical diagnostics. Ideally, it combines multiple imaging techniques to provide complementary anatomical and molecular information in living subjects. Particularly desirable are multimodal imaging probes capable of providing differential diagnostic signals upon interaction with specific molecular targets. Hydrogen peroxide (H2O2) is a key target in this regard, since it is typically overexpressed in cancer cells. In this study, we present a small-molecule probe that not only selectively detects endogenous H2O2 through multimodal imaging, with a significant H2O2-triggered 15-fold fluorescence enhancement but also turns “on” a chemical exchange saturation transfer (CEST) magnetic resonance (MR) response with 60-fold signal enhancement at pH 7.4. Excellent selectivity against various other biologically relevant species is seen. Using this probe, we observed 3.4–4.5-fold and 2.8–5.8-fold higher H2O2 levels in cancerous cell lines and tumor tissues compared to normal cell lines and tissues, respectively. Time-dependent in vivo fluorescence and CEST imaging in a HeLa (Henrietta Lacks) tumor xenograft mouse model revealed probe-dependent tumor detection by fluorescence and CEST MRI contrast in the tumor area. These observations are attributed to the relatively high endogenous H2O2 levels produced during mitosis. This newly developed probe holds promise for advancing our understanding of H2O2-related biology and for cancer detection both in vitro and in vivo.

A sensitive fluorescence biosensor based on ligation-transcription and CRISPR/Cas13a-assisted cascade amplification strategies to detect the H1N1 virus.

We propose a sensitive H1N1 virus fluorescence biosensor based on ligation-transcription and CRISPR/Cas13a-assisted cascade amplification strategies. Products are generated via the hybridization of single-stranded DNA (ssDNA) probes containing T7 promoter and crRNA templates to a target RNA sequence using SplintR ligase. This generates large crRNA quantities in the presence of T7 RNA polymerase. At such crRNA quantities, ternary Cas13a, crRNA, and activator complexes are successfully constructed and activate Cas13a to enhance fluorescence signal outputs. The biosensor sensitively and specifically monitored H1N1 viral RNA levels down to 3.23 pM and showed good linearity when H1N1 RNA concentrations were 100 pM-1 µM. Biosensor specificity was also excellent. Importantly, our biosensor may be used to detect other viral RNAs by altering the sequences of the two probe junctions, with potential applications for the clinical diagnosis of viruses and other biomedical studies.

Visualized Enzyme-Activated Fluorescence Probe for Accurately Detecting β-Gal in Living Cells and BALB/c Nude Mice.

Colorectal cancer was one of the major malignant tumors threatening human health and β-Gal was recognized as a principal biomarker for primary colorectal cancer. Thus, designing specific and efficient quantitative detection methods for measuring β-Gal enzyme activity was of great clinical test significance. Herein, an ultrasensitive detection method based on Turn-on fluorescence probe (CS-βGal) was reported for visualizing the detection of exogenous and endogenous β-galactosidase enzyme activity. The test method possessed a series of excellent performances, such as a significant fluorescence enhancement (about 11.3-fold), high selectivity as well as superior sensitivity. Furthermore, under the optimal experimental conditions, a relatively low limit of detection down to 0.024 U/mL was achieved for fluorescence titration experiment. It was thanks to the better biocompatibility and low cytotoxicity, CS-βGal had been triumphantly employed to visual detect endogenous and exogenous β-Gal concentration variations in living cells with noteworthy anti-interference performance. More biologically significant was the fact that the application of CS-βGal in BALB/c nude mice was also achieved successfully for monitoring endogenous β-Gal enzyme activity.

A dual-emission fluorescent probe for simultaneous detection of singlet oxygen and hypochlorous acid in lipid droplets

Singlet oxygen (1O2) and hypochlorite (ClO-) are two important reactive oxygen species. They coexist in living systems and can be interconverted in organisms, being involved in a variety of pathophysiological processes. Lipid droplets (LDs) are organelles that transport and store lipids, and 1O2 and ClO- tend to destroy unsaturated lipids, leading to lipid oxidation and causing a range of diseases. Here we proposed the first dual-response fluorescent probe DBCC that can simultaneously distinguish 1O2 and ClO- within LDs. The probe is almost non-fluorescent, while significant fluorescence enhancement can be observed in the blue and green channels after treatment with 1O2 and ClO-, respectively. With the excellent properties of high sensitivity, good selectivity and wide tolerance range, the probe DBCC has been successfully used to detect 1O2 and ClO- in HepG2 cells and zebrafish, and faultlessly realized endogenous and exogenous 1O2 and ClO- imaging in LDs. This work provides a powerful analytical tool for further investigating the levels of 1O2 and ClO- in LDs and associated diseases.

Development of D-π-A organic dyes for discriminating HSA from BSA and study on dye-HSA interaction

HSA (human serum albumin), a most abundant protein in blood serum, plays a key role in maintaining human health. Abnormal HSA level is correlated with many diseases, and thus has been used as an essential biomarker for therapeutic monitoring and biomedical diagnosis. Development of small-molecule fluorescent probes allowing the selective and sensitive recognition of HSA in in vitro and in vivo is of fundamental importance in basic biological research as well as medical diagnosis. Herein, we reported a series of new synthesized fluorescent dyes containing D-π-A constitution, which exhibited different optical properties in solution and solid state. Among them, dye M−H−SO3 with a hydrophilic sulfonate group at electron-acceptor part displayed selectivity for discrimination of HSA from BSA and other enzymes. Upon binding of dye M−H−SO3 with HSA, a significant fluorescence enhancement with a turn-on ratio about 96-fold was triggered. The detection limit was estimated to be ∼ 40 nM. Studies on the interaction mechanism revealed that dye M−H−SO3 could bind to site III of HSA with a 1:1 binding stoichiometry. Furthermore, dye M−H−SO3 has been applied to determine HSA in real urine samples with good recoveries, which provided a useful method for HSA analysis in biological fluids.

Development of Paper-Based Fluorescent Molecularly Imprinted Polymer Sensor for Rapid Detection of Lumpy Skin Disease Virus.

Lumpy Skin Disease (LSD) is a notifiable viral disease caused by Lumpy Skin Disease virus (LSDV). It is usually associated with high economic losses, including a loss of productivity, infertility, and death. LSDV shares genetic and antigenic similarities with Sheep pox virus (SPV) and Goat pox (GPV) virus. Hence, the LSDV traditional diagnostic tools faced many limitations regarding sensitivity, specificity, and cross-reactivity. Herein, we fabricated a paper-based turn-on fluorescent Molecularly Imprinted Polymer (MIP) sensor for the rapid detection of LSDV. The LSDV-MIPs sensor showed strong fluorescent intensity signal enhancement in response to the presence of the virus within minutes. Our sensor showed a limit of detection of 101 log10 TCID50/mL. Moreover, it showed significantly higher specificity to LSDV relative to other viruses, especially SPV. To our knowledge, this is the first record of a paper-based rapid detection test for LSDV depending on fluorescent turn-on behavior.

Rational design of a near-infrared fluorescent probe for monitoring butyrylcholinesterase activity and its application in development of inhibitors.

Butyrylcholinesterase (BChE) is widely expressed in multiple tissues and has a vital role in several key human disorders, such as Alzheimer's disease and tumorigenesis. However, the role of BChE in human disorders has not been investigated. Thus, to quantitatively detect and visualize dynamical variations in BChE activity is essential for exploring the biological roles of BChE in the progression of a number of human disorders. Herein, based on the substrate characteristics of BChE, we customized and synthesized three near-infrared (NIR) fluorescent probe substrates with cyanine-skeleton, and finally selected a NIR fluorescence probe substrate named CYBA. The CYBA demonstrated a significant increase in fluorescence when interacting with BChE, but mainly avoided AChE. Upon the addition of BChE, CYBA could be specifically hydrolyzed to TBO, resulting in a significant NIR fluorescence signal enhancement at 710nm. Systematic evaluation revealed that CYBA exhibited exceptional chemical stability in complex biosamples and possessed remarkable selectivity and sensitivity towards BChE. Moreover, CYBA was successfully applied for real-time imaging of endogenous BChE activity in two types of nerve-related living cells. Additionally, CYBA demonstrated exceptional stability in the detection of complex biological samples in plasma recovery studies (97.51%-104.01%). Furthermore, CYBA was used to construct a high-throughput screening (HTS) method for BChE inhibitors using human plasma as the enzyme source. We evaluated inhibitory effects of a series of natural products and four flavonoids were identified as potent inhibitors of BChE. Collectively, CYBA can serve as a practical tool to track the changes of BChE activity in complicated biological environments due to its excellent capabilities.

Highly selective and sensitive detection of hyaluronic acid based on a pyrene-cored cationic aggregation-induced emission luminogen

An aggregation-induced emission luminogen (AIEgen) that is positively charged, compound 1, is synthesized by attaching a pyridyl cation to a pyrene core by use of a cyanoethylene bond. By employing compound 1, it is possible to identify hyaluronic acid (HA) by combining it with the negatively charged HA through electrostatic interactions. When combined with HA, compound 1 exhibits a 9-fold fluorescence enhancement within 7 s, and shows a sensitive and quick response to HA with a detection limit as low as 6.9 ng/mL. Among the variety of available biological substances, even analogues to HA such as chondroitin 4-sulfate (CS) and heparin (Hep), compound 1 is shown to have the most significant fluorescence enhancement when connected to HA only, which demonstrates its excellent selectivity for HA. In particular, upon long-wavelength emission close to 600 nm and an extensive shift (∼160 nm), compound 1 allows for a minimization of the auto-fluorescence interference from complex biological substances and creates more accurate outcomes. Finally, the successful application of compound 1 for the purpose of selective and sensitive detection of HA in living cells is shown to be possible. Notably, good linear relationships arise for a serum assay when the concentration of the applied HA is within the range of 0–8.0 μg/mL (R2 = 0.999), which suggests a possible clinical practice for quantifying HA since the clinical dosage level is covered by this range during both cardiovascular surgery and long-term care. Furthermore, our research offers a feasible and straightforward tool for detecting HA through electrostatic interactions.

Molecular rotor bearing naphthol hydrazone Schiff base for sensing viscosity changes in solutions and biosystems

Viscosity is an important parameter in cells since it is closely related to a few severe human diseases. The development of fluorescent probes that can detect changes in intracellular viscosity has become an effective tool for monitoring the intracellular microenvironment. In this work, an efficient Schiff base molecular rotor, NaForm, is designed and synthesized by covalently linking a naphthol hydrazine derivative with the methyl 4-formylbenzoate group for the optical detection of viscosity changes. By increasing the viscosity of solvents, a significant enhancement of fluorescence for the probe at the yellow-green channel is observed. The probe also exhibits the advantages of high sensitivity and large Stokes shift of 128 nm. Both theoretical calculations and spectroscopic measurements suggest that the inhibition of chemical bond rotation between the hydrazine and the benzene ring leads to such a profound turn-on response. The successful applications of the probe in HeLa cells and zebrafish sufficiently demonstrate the privilege of NaForm as a practical viscosity-dependant chemosensor in aqueous samples and organisms. This chemosensor holds great promise for studying intracellular viscosity effects in various biological contexts.

A turn-on fluorescent probe based on benzothiazole for H2S detection and its multiple applications in water samples and food spoilage

Hydrogen sulfide (H2S) is a common hazardous gas, which is associated with environmental water pollution and food spoilage. Herein, a turn-on benzothiazole-based fluorescent probe (BATD) was synthesized for detecting H2S and characterized by single crystal X-ray diffraction, NMR and MS. BATD exhibited enhanced fluorescence signal at 600 nm with a remarked Stokes shift (145 nm), rapid response (within 90 s) and sensitivity (limit of detection, LOD = 76 nM) for H2S detection. The probe was significantly fluorescent enhancement in response to H2S over the other analytes. In addition, BATD was employed to detect H2S in water samples and monitor H2S gas generated from food spoilage. Benefiting from the excellent performance of the probe in recognizing H2S, a convenient H2S sensing platform for smartphones had been successfully established.

Construction of an Enzymatically Controlled DNA Nanomachine for One-Step Imaging of Telomerase in Living Cells.

Telomerase is a basic reverse transcriptase that maintains the telomere length in cells, and accurate and specific sensing of telomerase in living cells is critical for medical diagnostics and disease therapeutics. Herein, we demonstrate for the first time the construction of an enzymatically controlled DNA nanomachine with endogenous apurinic/apyrimidinic endonuclease 1 (APE1) as a driving force for one-step imaging of telomerase in living cells. The DNA nanomachine is designed by rational engineering of substrate probes and reporter probes embedded with an enzyme-activatable site (i.e., AP site) and their subsequent assembly on a gold nanoparticle (AuNP). Upon recognition and cleavage of the AP site in the substrate probe by APE1, the loop of the substrate probe unfolds, exposing telomeric primer (TP) with the 3'-OH end. Subsequently, the TP is elongated by telomerase at the 3'-OH end to generate a long telomeric product. The resultant telomeric product acts as a swing arm that can hybridize with a reporter probe to initiate the APE1-powered walking reaction, ultimately generating a significantly enhanced fluorescence signal. Notably, endogenous APE1 is used as the driving force of the DNA nanomachine, avoiding the introduction of exogenous auxiliary cofactors into the cellular microenvironment. Owing to the high kinetics and high amplification efficiency of the APE1-powered DNA nanomachine, this strategy enables one-step sensitive sensing of telomerase in vitro and in vivo. It can successfully discriminate telomerase activity between cancer cells and normal cells, screen telomerase inhibitors, and monitor the variations of telomerase activity in living cells, offering a prospective platform for molecular diagnostics and drug discovery.

Clusterization-triggered emission, stimulus-responsiveness, and DNA polyplex formation of poly(vinylamine)-based chiral block and random copolymers

Random and block copolymers exhibiting clusterization-triggered emission (CTE) and consisting of vinylamine (VAm) and methylated N-acryloyl-L-threonine (AThrOMe) were developed. They showed unique stimulus-responsiveness and polyplex-formation-induced changes in emission properties. The random and block copolymers, P(VAm-co-AThrOMe) and PVAm-b-PAThrOMe, contained no traditional fluorescent aromatic molecules, whereas the aggregation of VAm units within the assembled structures appeared to be a powerful tool for achieving enhanced fluorescence signals. The concentration-, wavelength-, and solvent-polarity-dependent emission behaviors of the VAm/AThrOMe copolymers could be tuned by implementing a rational molecular design (random or block sequence). The assembled structures and chiroptical and emission properties in response to external stimuli (temperature change and addition of salt and urea) in an aqueous solution were investigated using dynamic light scattering, circular dichroism, and fluorescence measurements. Selective interactions of DNAs with the VAm units in the VAm/AThrOMe copolymers were evaluated by performing agarose gel electrophoresis; these interactions affected the assembled structures and chiroptical and CTE properties. The comonomer sequence, external stimuli, and interactions with DNAs affected the aggregated states of the VAm unit in the copolymers, thereby showing that CTE behaviors can be manipulated in future sensing applications.

Zinc-Induced Luminescent l-Valine-Based Pseudopeptidic Soft Bioinspired Materials for Precise Tuning of Nanoassembly.

Pseudopeptide-based bioinspired materials are emerging for selective recognition of biologically significant analytes and are applicable in the modern nanoscience field. Therefore, we have developed novel multifunctional C2-symmetric soft pseudopeptides by amino acid l-valine and salicylaldehyde fragments using a series of aliphatic linkers. They are highly selective and sensitive to Zn (II) ions under physiological conditions and reveal significant fluorescence enhancement with the PET mechanism. The molecular self-assembly shows zinc-induced morphological transformation of the rod-shaped assembly into a chain-like morphology. Such a metal-induced hierarchical nano-assembly may have relevance for specific nanobiotechnology applications.

A novel isophorone-based NIR fluorescent and colormetric probe for Al3+ sensing and its application for living cells and plants imaging

In this paper, an isophorone-based NIR fluorescent and colormetric probe BDDH for Al3+ was synthesized and characterized, it showed highly selectivity and sensitivity through significant fluorescence enhancement and visible color change towards Al3+. The job plot confirmed that the binding ratio of BDDH with Al3+ was 1:1. Furthermore, the limit of detection (LOD) of Al3+ was determined to be 4.01 × 10−8 M. Moreover, BDDH was successfully applicated in identification of Al3+ in the different water samples, cell imaging in alive MCF-7 cells and plant imaging in soybean roots.

Construction of a label-free fluorescent biosensor for homogeneous detection of m6A eraser FTO in breast cancer tissues

Fat mass and obesity-associated protein (FTO) is a crucial eraser of RNA N6- methyladenosine (m6A) modification, and abnormal FTO expression level is implicated in pathogenesis of numerous cancers. Herein, we demonstrate the construction of a label-free fluorescent biosensor for homogeneous detection of m6A eraser FTO in breast cancer tissues. When FTO is present, it specifically erases the methyl group in m6A, inducing the cleavage of demethylated DNA by endonuclease DpnII and the generation of a single-stranded DNA (ssDNA) with a 3′-hydroxyl group. Subsequently, terminal deoxynucleotidyl transferase (TdT) promotes the incorporation of dTTPs into the ssDNA to obtain a long polythymidine (T) DNA sequence. The resultant long poly (T) DNA sequence can act as a template to trigger hyperbranched strand displacement amplification (HSDA), yielding numerous DNA fragments that may be stained by SYBR Gold to produce an enhanced fluorescence signal. This biosensor processes ultrahigh sensitivity with a detection limit of 1.65 × 10−10 mg/mL (2.6 fM), and it can detect the FTO activity in a single MCF-7 cell. Moreover, this biosensor can screen the FTO inhibitors, evaluate enzyme kinetic parameters, and discriminate the FTO expression levels in the tissues of breast cancer patients and healthy persons.

Development of a Dual Fluorescence Signal-Enhancement Immunosensor Based on Substrate Modification for Simultaneous Detection of Interleukin-6 and Procalcitonin.

High-sensitivity detection of biomarkers is of great significance to improve the accuracy of disease diagnosis and the rate of occult disease diagnosis. Using a substrate modification and two-color quantum dot (QD) nanobeads (QBs), we have developed a dual fluorescence signal-enhancement immunosensor for sensitive, simultaneous detection of interleukin 6 (IL-6) and procalcitonin (PCT) at low volumes (∼20 μL). First, the QBs compatible with QDs with different surface ligands were prepared by optimizing surfactants based on the microemulsion method. Through the use of a fluorescence-linked immunosorbent assay (FLISA), the feasibility of a dual signal-enhancement immunosensor was verified, and a 5-fold enhancement of fluorescence intensity was achieved after the directional coating of the antibodies on sulfhydryl functionalization (-SH) substrates and the preparation of QBs by using a polymer and silica double-protection method. Next, a simple polydimethylsiloxane (HS-PDMS) immunosensor with a low volume consumption was prepared. Under optimal conditions, we achieved the simultaneous detection of IL-6 and PCT with a linear range of 0.05-50 ng/mL, and the limit of detection (LOD) was 24 and 32 pg/mL, respectively. The result is comparable to two-color QBs-FLISA with a sulfhydryl microplate, even though only 20% of its volume was used. Thus, the dual fluorescence signal-enhancement HS-PDMS immunosensor offers the capability of early microvolume diagnosis of diseases, while the detection of inflammatory factors is clinically important for assisting disease diagnosis and determining disease progression.

Enhanced Efficiency of Pd(0)-Based Single Chain Polymeric Nanoparticles for in Vitro Prodrug Activation by Modulating the Polymer's Microstructure.

Bioorthogonal catalysis employing transition metal catalysts is a promising strategy for the in situ synthesis of imaging and therapeutic agents in biological environments. The transition metal Pd has been widely used as a bioorthogonal catalyst, but bare Pd poses challenges in water solubility and catalyst stability in cellular environments. In this work, Pd(0) loaded amphiphilic polymeric nanoparticles are applied to shield Pd in the presence of living cells for the in situ generation of a fluorescent dye and anticancer drugs. Pd(0) loaded polymeric nanoparticles prepared by the reduction of the corresponding Pd(II)-polymeric nanoparticles are highly active in the deprotection of pro-rhodamine dye and anticancer prodrugs, giving significant fluorescence enhancement and toxigenic effects, respectively, in HepG2 cells. In addition, we show that the microstructure of the polymeric nanoparticles for scaffolding Pd plays a critical role in tuning the catalytic efficiency, with the use of the ligand triphenylphosphine as a key factor for improving the catalyst stability in biological environments.