Fluorescence Turn-on Assay Research Articles

A turn-on fluorescence assay of tyrosinase activity based on an enzyme-triggered hydroxylation of phenol for conformation of silicon nanoparticles

Evolution of fluorometric sensing methods has brought an enormous opportunity and exhibited novel potentials for enzymatic activities determination. Herein, we have established an accurate and straightforward fluorescent sensing approach for tyrosinase (TYR) activity based on the formation of fluorescent silicon nanoparticles (Si NPs). Influenced by TYR-induced hydroxylation of phenol into catechol, and an easy reaction between catechol and 3(2-aminoethylamino) propyl (dimethoxymethylsilane) (AEAPDMMS) to generate Si NPs, we have designed a label-free fluorescence turn-on probe for TYR activity measurement. After optimization of all reaction parameters, the proposed approach could selectively determine TYR activity in the linearity range of 0.08–9 U mL−1 with the limit of detection (LOD) of 0.03 U mL−1. The practicability of the method in real sample was assessed by measuring TYR activity in human serum sample with successful results. In addition, Si NPs were wonderfully acceptable for Hela and SiHa cells imaging by virtue of their tolerable cytotoxicity and impressive biocompatibility. Therefore, the designed fluorescent biosensor exhibited a potential and widespread implementation in clinical diagnoses assays.

Characterization of RNA G-quadruplexes in porcine epidemic diarrhea virus genome and the antiviral activity of G-quadruplex ligands

Porcine epidemic diarrhea virus (PEDV), an enteropathogenic coronavirus, has catastrophic impacts on the global pig industry. However, there are still no anti-PEDV drugs with accurate targets. G-quadruplexes (G4s) are non-canonical secondary structures formed within guanine-rich regions of DNA or RNA, and have attracted great attention as potential targets for antiviral strategy. In this study, we reported two putative G4-forming sequences (PQS) in S and Nsp5 genes of PEDV genome based on bioinformatic analysis, and identified that S-PQS and Nsp5-PQS were enabled to fold into G4 structure by using circular dichroism spectroscopy and fluorescence turn-on assay. Furthermore, we verified that both S-PQS and Nsp5-PQS PQS could form G4 structure in live cells by immunofluorescence microscopy. In addition, G4-specific compounds, such as TMPyP4 and PDS, could significantly inhibit transcription, translation and proliferation of PEDV in vitro. Importantly, these compounds exert antiviral activity at the post-entry step of PEDV infection cycle, by inhibiting viral genome replication and protein expression. Lastly, we demonstrated that TMPyP4 can inhibit reporter gene expression by targeting G4 structure in Nsp5. Taken together, these findings not only reinforce the presence of viral G-quadruplex sequences in PEDV genome but also provide new insights into developing novel antiviral drugs targeting PEDV RNA G-quadruplexes.

Nucleotide-Based Lanthanide Coordination Polymer Nano-Probe for Turn-On Fluorescence Sensing of Zn2+ in Serum.

Water-dispersed lanthanide coordination polymers (LCPs) have attracted considerable attention owing to their superiority in bioanalysis. However, so far, most of the reported LCPs, due to the employment of water-insoluble and toxic organic molecules as ligands, are only competent in organic solution or the gaseous phase. Therefore, the construction of a water-dispersed, LCP-based, especially LCP nanoparticle (LCPNP)-based, sensor is still lacking and challenging. The aim was to obtain a novel and effective LCPNP-based sensor for Zn2+ by simple self-assembly, utilizing water-soluble guanosine monophosphate (GMP) as ligand and Eu3+ as luminescence center, . In aqueous solutions, Eu-GMP NPs were formed via self-assembly reaction between Eu3+ and GMP, and displayed very weak fluorescence due to low energy transfer from GMP to Eu3+ and the rate constant of nonradiactive deactivation of the excited states caused by the O-H vibration of coordinated water molecules. After the introduce of Zn2+, forming Eu-GMP/Zn, very interestingly, an 8-fold fluorescence enhancement was observed due to the removal of coordination water molecules and fluorescence sensitization of Zn2+. The fluorescence intensity of Eu-GMP NPs at 614 nm showed a linear relationship with the concentration of Zn2+ from 4 to 240 μM with a detection limit of 4 μM. Due to possessing long fluorescence, Eu-GMP showed prominent achievment for application in serum Zn2+ determination. The LCPNP probe exhibited excellent performance for the determination of Zn2+ in serum. For the first time, we developed and designed a kind of water-dispersed, LCPNP-based turn-on fluorescence assay for Zn2+ in serum. High sensitivity and good recoveries were achieved due to long fluorescence life, good water-dispersed behavior, and the turn-on fluorescence response of the LCPNP probe.

Dual mode assay of glutathione with Tb-doped g-C3N4/MnO2 nanoconjugates as fluorescence probe and Mn as elemental target

Glutathione (GSH) plays important roles in various physiological processes, thus highly sensitive assay of GSH and timely warning of its variation at trace level in complex biological matrixes is of great significance. However, this is challenging due to the coexisting reductive biomolecules and dynamic change of GSH levels in responding to various stimuli which remain largely unexploited. Herein, we report a dual mode protocol for the assay of GSH based on nanoconjugate g-C3N4:Tb/MnO2 between MnO2 nanosheets and terbium-doped g-C3N4 (g-C3N4:Tb) nanosheets. MnO2 moiety effectively quenches the emission at 546 nm from Tb3+ in the nanoconjugate, which is restored under the reduction of MnO2 by GSH to ensure fluorescence turn-on assay of GSH. Meanwhile, the generated Mn2+ facilitates inductively coupled plasma mass spectrometry (ICP-MS) detection to endow indirect highly sensitive assay of GSH. Fluorescence mode derived a limit of detection (LOD) of 0.17 μmol L−1 within a linear range of 0.5–160 μmol L−1, while ICP-MS resulted in a superior LOD of 0.016 μmol L−1 within 0.05–160 μmol L−1. Both detection modes provide excellent selectivity to GSH. The dual mode platform was validated by GSH assay in cell lysates. It was further demonstrated by monitoring the variation of dynamic change of GSH level under CuSO4 or cisplatin induced GSH consumption.

Label-free triplex DNA-based biosensing of transcription factor using fluorescence resonance energy transfer between N-doped carbon dot and gold nanoparticle

Herein, a new turn-on fluorescent assay was established as a platform for the sensing of transcription factor NF-kB p50 based on triplex DNA labeled with N-doped carbon dots (NCDs) and gold nanoparticles (AuNPs) as donors and acceptors, respectively in the fluorescence resonance energy transfer (FRET) system. The synthetized nanoparticles were studied by different characterization techniques. A labeled DNA molecule was designed to form a triplex when no target protein existence and reported its formation by the change in FRET efficiency. While the triplex DNA was formed, the fluorescence of carbon dots at 503 nm (excitation at 460 nm) was quenched by FRET between NCD and AuNP. However, presence of NF-kB p50 followed by the considerable enhancement in the fluorescence intensity caused by the release of AuNPs labeled single stranded DNA from the triplex DNA structure, used for sensitive determination of the transcription factor. This technique showed a linearity (R2 = 0.9943) in the range of 20–150 pM with a limit of detection of 9 pM for the determination of NF-kB p50. Moreover, the sequence-specific triplex-based biosensor could discriminate NF-kB p50 from the other proteins with high selectively. Our results suggest that the biosensor provides a generalizable platform for rapid detection of NF-kB p50 in synthetic medium, promising in prevention and early diagnosis of cancer.

A turn-on red-emitting fluorescent probe for determination of copper(II) ions in food samples and living zebrafish

Herein, we developed a turn-on red-emitting fluorescent probe for the sensitive and selective detection of copper ions (Cu2+) in food samples and living zebrafish. The probe employs a hemicyanine scaffold as the fluorophore and a 2-pyridinecarbonyl group as the recognition receptor and quenching moiety. The 2-pyridinecarbonyl moiety can be specifically cleaved by Cu2+ and results in an approximately 18-fold fluorescence enhancement of the probe, thereby providing a fluorescence turn-on assay for Cu2+. Additionally, the probe exhibited excellent selectivity, high sensitivity, a broad linear relationship (0.020 to 8.0 μM), and a low limit of detection (4.0 nM, S/N = 3) for Cu2+. Concomitantly, the probe exhibited satisfactory analytical performance when used with actual food samples. Moreover, the probe could be used for in situ determination of Cu2+ in both living plant tissues and in living zebrafish.

A turn-on fluorescent assay for glucose detection based on carbon dots/manganese dioxide assembly

In this work, a turn-on fluorescent assay based on carbon dots/manganese dioxide nanosheet (CDs/MnO2) assembly was developed for the detection of glucose. The fluorescence of CDs was first quenched by the addition of MnO2 nanosheet to form CDs/MnO2 assembly, which leads to a fluorescence resonance energy transfer (FRET) process from CDs to MnO2 nanosheet. When H2O2 was introduced into the quenched CDs solution, the MnO2 nanosheet was destroyed due to the redox reaction between H2O2 and MnO2 nanosheet, and the fluorescence of CDs was recovered accordingly. On this basis, by coupling with the oxidation reaction of glucose catalyzed by glucose oxidase, which produces gluconic acid and H2O2, a fluorescent assay for the quantitative analysis of glucose can be proposed. Under optimal conditions, the fluorescent intensity of CDs increased linearly with increasing concentrations of glucose from 2 to 200 μM, and the detection limit towards glucose was calculated to be 0.83 μM. In addition, the proposed fluorescent assay was successfully applied to the determination of glucose in serum samples with satisfactory results. This proposed fluorescent assay is simple, cost-effective and environmentally friendly, and thereby holding great potential for diabetes management and biosensing applications.

A new fluorescent probe for sensing of biothiols and screening of acetylcholinesterase inhibitors.

A new N2O-type BODIPY probe (LF-Bop) has been proposed for the selective and sensitive detection of biologically relevant small molecular thiols. This detection is based on the Michael addition reaction between the thiol and nitrostyrene groups in the probe, which decreases the quenching effect from the nitro group, thus resulting in the recovery of the deep-red fluorescence from the BODIPY structure. The results show that LF-Bop is able to detect all tested free thiols through a fluorescence turn-on assay. The lowest limit of detection (LOD) for glutathione was found to be down to nanomolar levels (220 nM). Based on this probe, we have developed a new fluorescence assay for the screening of acetylcholinesterase inhibitors. In total, 11 natural and synthetic alkaloids have been evaluated. Both experimental measurements and theoretical molecular docking results reveal that both natural berberine and its synthetic derivative dihydroberberine are potential inhibitors of acetylcholinesterase.

A fluorescence turn-on assay for simple and sensitive determination of hemin and blood stains

Hemin, a sort of important Fe(III)-coordinated protoporphyrin IX complexes, plays critical roles in many biological processes. Besides, hemin and its derivatives have been considered as biochemical markers to distinguish blood against other colored samples. The development of simple and sensitive methods for hemin determination is thus becoming important. In this research, a novel dihydrofluorescein derivative has been synthesized and applied as the fluorescence reporter. Inorganic H2O2 and organic tert-butyl hydroperoxide (TBHP), artemisinin (ART) have been used as peroxide sources to generate reactive radical species under hemin catalysis, which subsequently oxidize non fluorescent dihydrofluorescein to its fluorescent form. The limit of detection (LOD) was found down to picomolar levels (64 pM, 350 pM, 420 pM for H2O2, TBHP, ART systems respectively). Moreover, a simple detection strip has been prepared by incorporating both ART and fluorescence reporter into the filter papers, which offers a simple strategy to conveniently distinguish blood stains against other colored stains.

A turn-on fluorescence assay of alkaline phosphatase activity based on an enzyme-triggered conformational switch of G-quadruplex

Herein, a turn-on fluorescence assay was introduced for alkaline phosphatase (ALP) detection based on ThT/G-quadruplex system. The basis of the method is that chelation of guanine bases at the binding sites by Ag+ blocks G-quadruplex formation and decreases the fluorescence intensity sharply. In the presence of ALP, ascorbic acid 2-phosphate (AAP) is hydrolyzed to form ascorbic acid (AA) which in turn reduces Ag+ to Ag0. As a result, the blockage ability of Ag+ is disrupted which augments the fluorescence intensity and relies on the concentration of ALP. Under the optimized parameters (500 nM DNA probe; 6 μM Ag+; 1 mM AAP; 30 min for Ag+ and DNA probe reaction time), fluorescence intensity correlates linear range between 1 and 100 U/L of ALP concentration with the detection limit of 0.503 U/L. In the inhibition assay, 50% of ALP inhibition is caused by Na3VO4 with a concentration of 0.254 mM. Furthermore, the assay was used to detect ALP activity in human serum samples in which the results were significant. Above all, the proposed strategy is potential, facile, and sensitive for analyzing ALP activity and screening ALP inhibitor.

Turn-on fluorescent assay for antioxidants based on their inhibiting polymerization of dopamine on graphene quantum dots.

We describe a sensitive turn-on fluorescent assay for antioxidants by using fluorescence-tunable graphene quantum dots (GQDs). GQDs exhibited strong fluorescence without dopamine (DA). DA could self-polymerize to a thin polydopamine (PDA) film on the surface of GQDs under alkaline environment, resulting in the fluorescence quenching of GQDs via fluorescence resonance energy transfer (FRET). However, the self-polymerization of DA could be effectively inhibited in the presence of antioxidants including glutathione (GSH), ascorbic acid (AA), cysteine (Cys), and homocysteine (Hcys). Thus, the fluorescence of GQDs restored. The "turn-on" sensing of antioxidants could be achieved with high sensitivity. The detection limit for GSH, AA, Cys, and Hcys could be achieved as low as 2.4 nM, 1.5 nM, 4.2 nM, and 4.4 nM, respectively. Finally, the GQDs@PDA system was applied for monitoring cerebral antioxidants in rat brain microdialysates. This work promises new opportunities to evaluate antioxidant capacity in physiological and pathological fields.

A Fluorescence Inner-Filter Effect Based Sensing Platform for Turn-On Detection of Glutathione in Human Serum.

A novel turn-on fluorescence assay was developed for the rapid detection of glutathione (GSH) based on the inner-filter effect (IFE) and redox reaction. Molybdenum disulfide quantum dots (MoS2 QDs), which have stable fluorescent properties, were synthesized with hydrothermal method. Manganese dioxide nanosheets (MnO2 NSs) were prepared by exfoliating the bulk δ-MnO2 material in bovine serum albumin (BSA) aqueous solution. The morphology structures of the prepared nanoparticles were characterized by transmission electron microscope (TEM). Studies have shown that the fluorescence of MoS2 QDs could be quenched in the presence of MnO2 NSs as a result of the IFE, and is recovered after the addition of GSH to dissolve the MnO2 NSs. The fluorescence intensity showed a good linear relationship with the GSH concentration in the range 20–2500 μM, the limit of detection was 1.0 μM. The detection method was applied to the analysis of GSH in human serum samples. This simple, rapid, and cost-effective method has great potential in analyzing GSH and in disease diagnosis.

A label-free and fluorescence turn-on assay for sensitive detection of hyaluronidase based on hyaluronan-induced perylene self-assembly

A label-free and fluorescence turn-on assay for sensitive detection of HAase based on HA–PDI nanoaggregates was reported.

Investigation of the surface confinement effect of copper nanoclusters: construction of an ultrasensitive fluorescence turn-on bio-enzyme sensing platform.

Copper nanoclusters (CuNCs) have attracted considerable research interest due to their good physicochemical properties, ease of preparation, and low price. However, the low quantum yield and poor stability in aqueous solutions have greatly limited their applications. In order to improve the fluorescence properties and stability of CuNCs, in this paper, the surface confinement effect of CuNCs based on 2D layered double hydroxide (LDH) was proposed to prepare the fluorescent composites of glutathione protected CuNCs and LDH (GS-CuNCs/LDH) with excellent quantum yield and long fluorescence lifetime. Moreover, a novel, simple, and ultrasensitive fluorescence assay for the detection of hyaluronidase was proposed based on the surface confinement effect. The limit of detection for hyaluronidase was as low as 0.014 U mL-1. For the first time, this work developed a bio-enzyme sensing platform based on the surface confinement effect, which can serve as a promising candidate in biosensing.

Fluorescence turn-on assay for detection of serum D-penicillamine based on papain@AuNCs-Cu2+ complex

Papain-stabilized gold nanoclusters (papain@AuNCs) with a red fluorescence emission at 639 nm have been generated successfully in aqueous solution. The fluorescence of papain@AuNCs could be quenched in the addition of Cu2+. Subsequently, a unique fluorescent probe based on papain@AuNCs-Cu2+ complex has been constructed for sensitive, selective and “turn-on” detection of D-penicillamine (D-pen). The sensing probe has exhibited a remarkable fluorescence enhancement in the presence of D-pen, which can be measured ranging from 30.0 μM to 2.0 mM (r2 = 0.991) with the detection limit of 5.0 μM. Furthermore, the developed assay has been utilized to explore the metabolic process of D-pen in rats after intraperitoneal injection. As far as we concerned, the present study has reported the first analytical method for sensing serum D-pen in real time by using the fluorescent papain@AuNCs-Cu2+ complex. Our strategy paves a way for digging up the biological and clinical application of the fluorescent AuNCs.

Probing glutathione reductase activity with graphene quantum dots and gold nanoparticles system

Graphene quantum dots (GQDs) and gold nanoparticles (AuNPs) were combined for probing glutathione reductase (GR) activity. The main probing mechanism is based on the following facts: (1) The fluorescence of GQDs can be quenched by AuNPs through fluorescence resonance energy transfer (FRET). (2) Reduced glutathione (GSH) can protect AuNPs from being aggregated and enlarge the inter-particle distance between GQDs and AuNPs, which subsequently produces the fluorescent signal recovery. (3) AuNPs has much weaker affinity towards oxidized glutathione (GSSG) in contrast to GSH. (4) GR can catalyze the reduction of GSSG into GSH in the presence of coenzyme β-nicotinamide adenine dinucleotide 2′-phophate hydrate (NADPH). Based on these facts, a turn-on fluorescent assay for GR has thus been developed based on the GQDs-AuNPs system. The proposed assay can allow for the evaluation of GR activity in the range of 0.0050–0.13 mU/mL, with a minimum detectable concentration of 0.0050 mU/mL, which was more than one order of magnitude lower than most of the current sensing platforms. Besides, this GQDs-AuNPs based GR assay was successfully applied for screening the inhibitor of GR activity by using 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) as a model.

Synthesis of fluorescent molecularly imprinted nanoparticles for turn-on fluorescence assay using one-pot synthetic method and a preliminary microfluidic approach

Fluorescent molecularly imprinted polymers (MIPs) have received considerable attention for their promising applications in sensing, imaging and diagnostics. Different synthetic methods have been reported to prepare fluorescent MIPs. Here, one-pot polymerization method was developed to synthesize fluorescent MIP nanoparticles (NPs) bearing a suitable fluorescent label as the fluorescence acceptor. The MIP NPs can emit strong fluorescence based on fluorescence resonance energy transfer (FRET) when they bind to analytes acting as the fluorescence donor. The one-pot synthetic method is straightforward, less time-consuming and easy to optimize the loading of fluorescence acceptor. Additionally, a preliminary microfluidic reactor was designed for preparing such fluorescent MIP NPs with more uniform size in a continuous process for the first time. The fluorescent MIP NPs can be used for selective detection of the analytical target in a FRET-based turn-on fluorescence assay which is easy to carry out with no tedious separation steps.

Turn-on fluorescent assay based on purification system via magnetic separation for highly sensitive probing of adenosine

In this work, based on purifying the analysis system by magnetic separation, we constructed a turn-on fluorescent assay for highly sensitive detection of possible biomarkers, employing adenosine as the model target analyte. Aptamer labeled-carbon dots (Apt-CDs) was applied as fluorescence probes as well as selective recognition elements, while Fe3O4@polypyrrole (Fe3O4@PPY) was used as fluorescence quenching and magnetic separating materials for system purification. In absence of adenosine, the Apt-CDs were attached to the surface of PPY through π-π stacking and hydrophobic interaction and its fluorescence was quenched by Fe3O4@PPY. In presence of adenosine, the aptamer could specifically bind with adenosine to form a three dimensional Apt-CDs-adenosine complex and detach from PPY, leading to the fluorescence recovery of Apt-CDs. More importantly, with analysis system purification via twice convenient magnetic separations of free CDs and Apt-CDs-adenosine complex respectively, the detection limit could be reduced from 8 nmol L−1 to 0.15 nmol L−1. This concept offers potential application for simple, rapid, cost-effective, and highly sensitive assay of biological samples.

Hemoglobin-incorporated iron quantum clusters as a novel fluorometric and colorimetric probe for sensing and cellular imaging of Zn(II) and cysteine.

The authors describe a novel water-soluble, stable, biocompatible, and highly fluorescent probe consisting of iron quantum clusters incorporated into human adult hemoglobin (Hb-FeQCs). The Hb-FeQCs were characterized by various spectroscopic techniques. The probe displays strong absorption and yellow fluorescence with a peak centered at 567nm (photo-excited at 460nm). The Hb-FeQCs show excellent photostability over a wide range of pH values (5-12), even in the presence of high electrolyte concentrations. A colorimetric and a fluorometric method were worked out for the quantitation Zn(II) and cysteine in aqueous solution. Zinc ions induce a visible color change from brown to yellow. The sensitivity of Hb-FeQCs towards other metal ions was negligible, with the exception of Co2+ and Cu2+, which caused a modest interference. The Hb-FeQCs were exploited in a sensitive and selective turn-on fluorescence assay for Zn2+. It is also found that cysteine quenches the fluorescence of the Hb-FeQCs/Zn(II) complex. Under the optimized conditions, the probe has a linear response in the 0.04 to 2.2μM Zn(II) concentration range, with a 48nM detection limit. Response to cysteine is linear in the 1-60μM concentration range, with a 0.25μM limit of detection. This fluorescent probe undergoes fluorescent emission intensity enhancement upon binding to zinc ions in living normal human fibroblast cells under visible lamp. The cellular imaging capability and very low cytotoxicity of this soluble iron quantum clusters can be potentially extended as an exciting sub-nanoplatform with promising biomaterial applications. Graphical abstract Schematic of yellow-emitting iron quantum clusters in hemoglobin matrix (Hb-FeQCs) were characterized and successfully applied for sensing zinc(II) and cysteine. The act as an on-off fluorescent probe and can be appliedto image zinc ions in human fibroblast cells under visible light.

UV-light-induced synthesis of PEI-CuNCs based on Cu2+-quenched fluorescence turn-on assay for sensitive detection of biothiols, acetylcholinesterase activity and inhibitor

Polyethyleneimine-protected copper nanoclusters (PEI-CuNCs) were synthesized and applied to detect Cu2+, biothiols, and acetylcholinesterase (AChE). A facile UV-light-induced method was used in the synthesize process to obtain PEI-CuNCs with high stability and strong fluorescence intensity. Based on the quenching mechanism, a label-free assay for sensing Cu2+ by PEI-CuNCs was developed with the limit of detection (LOD) of 0.12 μM. Biothiols with SH functional groups could react with Cu2+ to form the RSH–Cu2+ complex, making Cu2+-triggered fluorescence quenching turned on. Two biothiols, glutathione and cysteine, were determined with LODs of 0.26 μM and 0.34 μM within linear ranges of 0.5–25 μM and 1–25 μM, respectively. AChE could hydrolyze acetylthiocholine into thiocholine (TCh), which reacted with Cu2+ to form TCh–Cu2+ complex and turned on Cu2+-quenched fluorescence. LOD of AChE was calculated to be 1.38 mU/mL within a linear range from 3 to 200 mU/mL. A typical inhibitor of AChE, tacrine, was also detected by this convenient fluorescence turn-on assay with the corresponding IC50 value (the inhibitor concentration required for 50% inhibition of the enzyme activity) of 53.4 nM. Moreover, the assay was successfully applied to the detection of biothiols and AChE activity in human serum samples. Results demonstrated the great potential of PEI-CuNCs and paved a new avenue for exploring their versatile biosensing applications.